Intercalators having affinity for DNA and methods of use

ABSTRACT

Intercalator compounds of formula I-T m  as defined herein are provided which are comprised of intercalator moieties, or substituted intercalator moieties, having one or more functionalized chains, or moieties, and which compounds provide a high affinity for binding to the DNA molecule and show reduced self-quenching, while providing superior transport kinetics. The compounds have been found to provide enhanced fluorescence when bound to a DNA molecule within a fluorescent flow cytometry environment which is about eight to ten times brighter in fluorescence than &#34;bis&#34; structure conventional intercalating agents and other known intercalating agents utilized in flow cytometry environment.

This application is a continuation of application Ser. No. 08/265,342,filed Jun. 23, 1994, abandoned which is a continuation-in-partapplication of U.S. Ser. No. 08/086,285 filed Jun. 30, 1993 abandoned.

BACKGROUND

The present invention relates to intercalator compounds, and the use ofsuch compounds, each of which is comprised of an intercalator moiety, ora substituted intercalator moiety, derivatized with one or morefunctionalized chains, or moieties, and which compounds have highaffinity for binding to a DNA molecule. These intercalator compoundsexhibit improved binding to a DNA molecule within known methodologiesrequiring intercalator insertion into the DNA molecule. Still, theinvention relates to enhanced binding of DNA molecules by anintercalator-functioning segment utilized in labeling, capture,therapeutic insertion, assay and the like, with improved performance ofthe intercalator due to the increased utilization efficiency of thecompounds.

The term "intercalator" was introduced into the chemistry field over 30years ago to describe the insertion of planar aromatic or heteroaromaticcompounds between adjacent base pairs of double stranded DNA (dsDNA).Many DNA intercalating compounds elicit biologically interestingproperties. It is generally agreed that these properties are related totheir reactivity with DNA. In the search for more active compounds, itis logical to design molecules with the highest possible affinity forDNA. In 1990, it was reported that complexes of ethidium homodimer withdsDNA performed at ratios of one dimer per four to five base pairs, andwere stable to electrophoresis on agarose gels. This allowedfluorescence, detection and quantitation of DNA fragments with picogramsensitivity after separation and complete absence of background stain.Such a result has been sought through various manipulations ofintercalator compounds, for example, by functional compounds made up ofDNA intercalating dyes. As a result of these efforts, DNA intercalatingagents utilizing ethidium bromide have been used in various DNAanalytical procedures.

Various reported DNA intercalating agents utilizing ethidium bromidehave been used in a multitude of DNA analytical procedures, for example:

Christen, et al., "An Ethidium Bromide-Agarose Plate Assay for theNonradioactive Detection of CDNA Synthesis", Anal. Biochem., 178 (2),May 1, 1989, pp. 269-272, report ethidium bromide was used to determinethe success of cDNA synthesis reactions. Since ethidium bromide inagarose can be used to quantitate RNA and DNA, conditions under whichthe greater fluorescence of double-stranded DNA is utilized were devisedto assay double stranded DNA synthesis from mRNA. Ethidium bromide at 5micrograms/ml in agarose allowed quantitative detection of cDNA in therange of 0.03 to 0.0015 microgram. Sodium dodecyl sulfate had an adverseeffect on the measurement of cDNA. Subsequent cDNA analysis by alkalinegel electrophoresis and staining in 5 micrograms/ml ethidium bromideallowed accurate and rapid sizing of cDNA and required only 0.01-0.05microgram cDNA.

Petersen, S. E., "Accuracy and Reliability of Flow Cytometry DNAAnalysis Using a Simple, One-Step Ethidium Bromide Staining Protocol",Cytometry, 7 (4), July, 1986, pp. 301-306, reports that sources ofvariation and error were investigated for a simple flow cytometricanalysis of DNA content of detergent-isolated nuclei stained withethidium bromide.

In "Ethidium Bromide in the Detection of Antibodies to DNA and ofCirculating DNA by Two-Stage Counterimmunoelectrophoresis", J. Immunol.Methods, 85 (1), Dec. 17, 1985, pp. 217-220, Riboldi, et al., reportthat in an attempt to overcome the limitations ofcounterimmunoelectrophoresis in the detection of precipitating anti-DNAantibodies or circulating DNA, ethidium bromide was used to increase thevisibility of the precipitating lines and to confirm their specificity.

W. A. Denny reported in "DNA-Intercalating Ligands as Anti-Cancer Drugs:Prospects for Future Design", Anticancer Drug Des., 4 (4), December,1989, pp. 241-263, that interest in DNA-intercalating ligands asanti-cancer drugs has developed greatly since the clinical success ofdoxsorubicin.

A number of agents have been described for labeling nucleic acids,whether probe or target, for facilitating detection of target nucleicacid. Suitable labels may provide signals detectable by fluorescence,radioactivity, colorimetry, X-ray diffraction or absorption, magnetismor enzymatic activity, and include, for example, fluorophores,chromophores, radioactive isotopes, enzymes, and ligands having specificbinding partners.

Fluorescent dyes are suitable for detecting nucleic acids. For example,ethidium bromide is an intercalating agent that displays increasedfluorescence when bound to double stranded DNA rather than when in freesolution. Ethidium bromide can be used to detect both single and doublestranded nucleic acids, although the affinity of ethidium bromide forsingle stranded nucleic acid is relatively low. Ethidium bromide isroutinely used to detect nucleic acids following gel electrophoresis.Following size fractionation on an approximate gel matrix, for example,agarose or acrylamide, the gel is soaked in a dilute solution ofethidium bromide.

The use of fluorescence labeled polynucleotide probes and polynucleotidehybridization assays have been reported. According to these methods,probes are prepared by attaching a particular absorber-emitter moietiesto the three prime and five prime ends of the nucleic acid fragments.The fragments are capable of hybridizing to adjacent positions of atarget DNA so that if both fragments are hybridized, the proximity ofthe absorber and emitter moieties results in the detectable emitterfluorescence. According to these methods, the fluorescent dye isintroduced into the target DNA after all in vitro nucleic acidpolymerizations have been completed. The inhibitory effects ofintercalating agents on nucleic acid polymerases have been described innumerous locations.

DNA binding dyes are useful as antibiotics because of the inhibitoryeffects of nucleic acid replication processes that result from the agentbinding to the template. The use of intercalating agents for blockinginfectivity of influenza or herpes viruses have been reported. It hasalso been reported and described that a number of DNA binding agents,both intercalators and nonintercalators, inhibit nucleic acidreplication. For example, ethidium bromide inhibits DNA replication.

Methods have been provided for detecting a target nucleic acid in asample. These methods comprise the steps of (a) providing an amplifiedreaction mixture that comprises a sample, a DNA binding agent, wheresaid agent is characterized by providing a detectable signal when boundto double stranded nucleic acid, which signal is distinguishable fromthe signal provided by said agent when it is unbound, and reagents foramplification; (b) determining the amount of signal produced by themixture of step (a); (c) treating said mixture under conditions foramplifying the target nucleic acid; (d) determining the amount of saidsignal produced by the mixture of step (c); and (e) determining ifamplification has occurred. These DNA binding intercalating agents, suchas ethidium bromide or ethidium homodimer allow fluorometric study ofthe interaction of various molecules with DNA.

The intercalating agent useful for DNA binding or detecting amplifiednucleic acids is an agent or moiety capable of insertion between stackedbase pairs in the nucleic acid double helix. Intercalating agents suchas ethidium homodimer and ethidium bromide fluoresce more intensely whenintercalated into double stranded DNA than when bound to single strandedDNA, RNA, or in solution. Other uses of intercalators have been in thefield of separation and isolation or purification of nucleic acids fromcomplex biological or clinical specimens.

Various methods of separating deoxyribonucleic acids (DNA) from liquidbiological samples are known in the art, but are very time consuming orotherwise plagued by complication. It is known that DNA adheres tonitrocellulose. The liquid sample containing DNA is applied to anitrocellulose filter and the DNA adheres or binds to the filter.

Another method of separating DNA from samples is ultracentrifugationwith sucrose or cesium chloride density gradients. The DNA is separatedfrom other macromolecules such as proteins by this method according tothe buoyant density or sedimentation coefficient. The biological sampleis layered onto the density gradient in a centrifuge tube and is spun atvery high speeds for long periods of time for DNA to travel through thedensity gradient. This method, although satisfactory, is very timeconsuming and labor intensive. The centrifugation time may be 20 hoursor more per sample. Furthermore, if the sample is spun too long, the DNAwill not only separate from the sample but also will pass entirelythrough the gradient to the very bottom of the centrifuge tube alongwith other constituents in the sample. Therefore, this method is alsonot suitable as a fast and easy method for separating DNA from complexsamples.

Agarose polyacrylamide gel electrophoresis is also used to separate DNAfrom biological samples. In this method, the sample is applied to oneend of a glass or plastic receptacle containing the gel and an electriccurrent is applied across the length of the receptacle. The negativelycharged nucleic acid molecules move toward the anode, the largermolecules moving more slowly. The rates of migration of the moleculesdepend on their molecular weights and on the concentration and degree ofcross linking in the gel material. The DNA is then removed from the gelby cutting out that portion of the gel in which the DNA is located andfinally extracting the DNA. Again, this method is time consuming andlabor intensive, and the DNA must still be separated from the gel. WhenDNA is separated by the electrophoresis gel method or by centrifugation,it is necessary for the DNA to be stained in some manner to bevisualized. Typically, ethidium bromide (EtBr) has been used as thestaining agent. Ethidium bromide adheres to the DNA by intercalationbetween the base pairs of the double helix structure of the DNA.

More recently, an ethidium homodimer has been synthesized and introducedwith bifunctional intercalators in order to allow fluorometric studyincluding the interaction of such molecules with DNA. It has beendetermined that the ethidium homodimer ("EthD") binds to double strandedDNA ("dsDNA") about two (2) orders of magnitude more strongly thanethidium bromide. Complexes of EthD with dsDNA have performed at a ratioof one dimer per 4 to 5 base pairs and were found to be stable toelectrophoresis on agarose base. On binding to dsDNA, the fluorescencequantum yield of the dimer increases 40 fold independent of nucleotidesequence.

Stable dsDNA-fluoropore complexes can be formed to obtain anywhere fromseveral to several thousand fluoropores each, as desired. Under suitablecontrolled conditions these complexes do not transfer dye to othernucleic acids or proteins. An important property of these complexes isthat their fluorescence emission intensity is a linear function of thenumber of intercalated dye molecules. As high sensitivity fluorescencedetection apparatus becomes more generally available, the ability to usedyes to replace, for example, radioactivity for sensitivity detection ofDNA, is becoming more and more valuable.

Dye dsDNA complexes represent a novel family of fluorescence labels witha wide range of spectroscopic properties whose composition, structureand size can be tailored to particular applications. DNA molecules canbe readily derivatized to attach biotin, digoxigenin or any number ofother substituents that can be recognized by avidin or antibodies. Suchderivatized DNA molecules loaded with dye may allow detection at muchhigher sensitivity in numerous applications, for example, immunoassay,fluorescence, and in situ hybridization of chromosomes and the like thatcurrently use other fluorescence labels.

Probes with a double stranded region, which provide intercalation sitesand a single stranded region to allow recognition by hybridization ofspecific target sequences, offer another approach to the generation ofversatile fluorescent labels. Development of conditions that allow cleardiscrimination between the binding of intercalators to single and doublestranded nucleic acids is an essential prerequisite to the use of suchprobes.

Fluorescent probes are valuable reagents for the analysis and separationof molecules and cells. Some specific examples of their application areidentification and separation from a subpopulation of cells in a mixtureof cells by the techniques of fluorescence, flow cytometry,fluorescence-activated cell sorting, and fluorescence microscopy. Otherapplications include determination of a concentration of a substance ormember of a specific binding pair that binds to a second species, ormember of the specific binding pair, e.g., antigen-antibody reactions inan immunofluorescent assay. Still another application is thelocalization of substance in gels and other insoluble supports by thetechniques of fluorescence staining.

Choice of fluorescers for these purposes is hampered by variousconstraints; one being the absorption and emission characteristics ofthe fluorescer since many ligands, receptors and other binding pairmembers, as well as other extraneous materials associated with thesample, for example, blood, urine and cerebrospinal fluid, willauto-fluoresce and interfere with an accurate determination orquantification of the fluorescent signal generated by the fluorescentlabel when the sample is exposed to the appropriate stimulus. Anotherconsideration is the quantum efficiency of the fluorescer. Yet anotherconcern is self-quenching; this can occur when the fluorescent moleculesinteract with each other when in close proximity. An additional concernis the non-specific binding of the fluorescer to other compounds or evenwith the test container.

It has been shown that dsDNA forms highly fluorescent complexes with thebis-intercalator EthD. Observations regarding the bis-intercalator EthDsuggest that the intercalator can be exploited to generate a family ofhighly fluorescent stable dsDNA-dye complexes with distinctiveproperties. Such complexes could be exploited by multiplex detection ofdsDNA fragments, as well as many analytical applications in whichappropriately diversified dsDNA fragments labeled noncovalently withdifferent dyes could be used as a unique family of fluorescent probes:##STR1## However, this compound may have a tendency to self-quench whenbound to DNA.

In flow cytometry apparatuses, cells or other particles are caused toflow in a liquid flow stream so as to facilitate the investigation ofcertain characteristics thereof. In general, a flow cytometry apparatusis useful for identifying the presence of certain cells or particles ofinterest, enumerating those cells or particles and, in some instances,providing a sorting capability so as to be able to collect those cellsor particles of interest. In a typical flow cytometry apparatus, a fluidsample containing cells is directed through the apparatus in a rapidlymoving liquid stream so that each cell passes serially, andsubstantially one at a time, through a sensing region. Cell volume maybe determined by changes in electrical impedance as each cell passesthrough the sensing region. Similarly, if an incident beam of light isdirected at the sensing region, the passing cells scatter such light asthey pass therethrough. This scattered light has served as a function ofcell shape and size, index of refraction, opacity, granularity,roughness and the like. Further, fluorescence emitted by labeled cells,or autofluorescent cells, which have been excited as a result of passingthrough the excitation energy of the incident light beam is detectablefor identification of cells having fluorescent properties. After cellanalysis is performed by the flow cytometry apparatus, those cells thathave been identified as having the desired properties may be sorted ifthe apparatus has been designed with such capability.

Instruments such as flow cytometry apparatuses are particularly usefulfor researchers and investigators studying various responses, reactionsand functions of the immune system. Immunofluorescence studies, as wellas fluorescence immunoassays, assist the investigator in identifying andtargeting select cells of interest so that disease states, conditionsand the like may be properly characterized. In addition to immune systeminvestigations, fluorescence analysis is also quite beneficial in cellbiology and morphology investigations, including the study of thesubstrate of cellular material.

In relying upon fluorescence to provide data and information aboutcells, the mechanics of performing tests for the fluorescence responseis a major consideration in the design of the instrument as well as theresults obtained. Specifically, the fluorescent markers, whether suchmarkers be fluorescent stains or dyes, are typically excited by lightenergy. Usually there is an optimal wavelength which provides thegreatest level of excitation for the fluorochromatic marker being used.Once excited, fluorescence emission occurs typically at wavelengthsdifferent from the wavelength of excitation. Fluorescence analysisinstruments, whether fluorescence microscopes, image analyzers or flowcytometers, are generally designed to detect the fluorescence emissionat the wavelength of emission maxima where the fluorescence signal isstrongest.

Before the discovery and publication of the utilities of ethidiumhomodimer as an important intercalator, the usual intercalator of choicewas ethidium bromide. Uses of the ethidium bromide intercalators includefluorometric methodologies, quantitative fluorescences of DNAintercalated ethidium bromide on agarose gels, ethidium bromide-agaroseplate assay or detection of false DNA analysis and the like. Ethidiumbromide and propidium bromide were further used in flow cytometry, aswell as applications for direct electronic imaging, direct and rapidquantitation of fluorescence and electrophoretic gels in application asethidium bromide-stain DNA. Ethidium bromide has also been used toincrease the visibility of the precipitant lines and to confirm thespecificity in two stage counter immunoelectrophoresis methodologies fordetection of participating anti-DNA antibodies or circulating DNA.Utilization of ethidium bromide as an intercalator in numerousenvironments, as well as the more recent utilization of the ethidiumhomodimer intercalator are well documented in the literature and presentthe leading edge of intercalator methodology and efficiency.

In a somewhat different application of ethidium bromide as a stainingagent, ethidium bromide has been linked to a solid support. U.S. Pat.No. 4,119,521, issued to Chirikjian on Oct. 10, 1978, discloses afluorescent DNA intercalating agent derivative of activatedpolysaccharides. The derivatives in the patent function as fluorescentstains to provide direct visualization of the DNA and their fractions,under the excitation of shortwave, ultraviolet radiation. Theintercalating agents used in the patent are ethidium halides, with thepreferred agent being ethidium bromide. This agent is coupled covalentlyto an activated polysaccharide such as agarose.

Utilization of ethidium bromide as an intercalator for use in numerousenvironments, as well as the more recent utilization of the ethidiumhomodimer intercalator are well documented in the literature and presentthe leading edge of intercalator methodology and efficiency. However,there remains an ever present need to improve utilization of theintercalators and viability of the use of intercalators with DNA,specifically addressing (1) high affinity for binding the intercalatorsto the DNA molecule; (2) reduction of self-quenching; and (3) providingsuperior transport kinetics. Intercalators possessing these qualitiesreduce the amount of intercalator required for performing one of themany functions involved in the aforementioned methodologies which canalso enhance methodologies. In addition, improvement in accuracy andreliability of the various uses of interest is of continuing concern.

SUMMARY

Broadly, the invention provides a compound having an "I" moiety bondedto one or more "T" moiety. The general formula of the compounds arerepresented by:

I-(T)_(m)

wherein the I moiety denotes an aromatic or heteroaromatic segment; theT moiety denotes a "tail" or "chain" moiety; and m is an integer from 1to 5. When m is more than 1, T can be similar or different from oneanother.

In one aspect, the present invention provides a compound having an Imoiety bonded to one or more T moiety, the T moieties, when more thanone T moiety are present, are the same or different from one another,the T moiety having the formula of: ##STR2## and the compound having aformula: ##STR3## wherein: I is an aromatic or heteroaromatic segment;

X is a heteroatom selected from the group consisting of nitrogen andsulfur;

R, R₁ and R₂ are the same or different from one another and are alkyl,alicyclic, heteroalicyclic, aromatic or heteroaromatic groups;

R₃ and R₄ are hydrogen when X is nitrogen; R₃ and R₄ are methyl, ethyl,or phenyl groups when X is sulfur;

k is zero or an integer from 1 to 10;

q is zero or an integer from 1 to 10;

n is an integer from 20 to 20;

m is an integer from 1 to 5;

o is zero or one; and

p is zero or one;

and the acid addition salts thereof;

provided that the I moiety is not phenanthridinium when: X is nitrogen;R₃ and R₄ are hydrogens R, R₁ and R₂ are methylene groups; o is 1; p is1; m is 1; and the acid addition salts thereof;

provided that the I moiety is not phenanthridinium when: X is nitrogen;R₃ and R₄ are hydrogens; R, R₁ and R₂ are methylene groups; o is 1; p is1; m is 2; a first T moiety directly bonded to the I moiety in which nis 2, k is 1 and q is zero; a second T moiety, directly bonded to thefirst T moiety, in which n is 2, and k and q are zero; and acid additionsalts thereof;

provided that the I moiety is not phenanthridinium when: X is nitrogen;R₃ and R₄ are hydrogens; R, R₁ and R₂ are methylene groups; o is 1; p is1; m is 2; a first T moiety directly bonded to said I moiety in which nis 2, k is 1 and q is zero; a second T moiety, directly bonded to thefirst T moiety, in which n is 2, k is 1 and q is 1 and acid additionsalts thereof; and

provided that the I moiety is not phenanthridinium when: X is nitrogen;R₃ and R₄ are hydrogen; o is 1; p is 1; m is 3; n is 2; k is 1; q iszero; and acid addition salts thereof.

The invention provides compounds comprised of intercalator moieties orsubstituted intercalator moieties having a functionalized chain, whichcompounds provide a high affinity for binding to the DNA molecule andshow reduced self-quenching while providing superior transport kinetics.The inventive intercalators have been found to provide enhancedfluorescence when bound to a DNA molecule within a fluorescent flowcytometry environment which is about eight to ten times brighter influorescence than ethidium homodimer utilized in the same flow cytometryenvironment. Because of the enhancement of fluorescence, the detectionof DNA hybridization can be accomplished using much lower concentrationsof intercalator compounds of the present invention than usingconventional intercalating agents, such as ethidium homodimer orethidium bromide. Using the same concentrations, intercalator compoundsof the present invention can detect far less amounts of DNAhybridization than can conventional intercalating agents. Thus, theintercalator compounds of the present invention are far more sensitivethan the known intercalating agents in detecting DNA hybridization.

Improvements in flow cytometry, fluorescence in-situ hybridizationassays, gel electrophoresis, DNA detection, immunoassay for DNA, andother DNA studies are substantial. The use of intercalators with DNA andmultiple methods have shown that the ethidium homodimer is about twoorders of magnitude brighter than conventional staining methodology,i.e., ethidium bromide. However, the intercalator compounds inaccordance with the present invention provide, for example, a dye whichexhibits an eight to ten-fold increase in brightness over that of EthDin the same environment, or about a thousandfold improvement over moreconventional staining methodologies. With pre-staining andpost-electrophoresis detection, the sensitivity level ofradioimmunoassay detection of DNA is now attainable with fluorophores.The compound compositions provided by this invention extends the limitsof detection by up to tenfold over EthD, thereby providing a potentialfor new uses in applications of intercalators for the study of DNAanalysis, as well as therapeutics and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a FACScan™ display for side scatter versus forward scatter;

FIG. 2 is a histogram of fluorescence intensity (abscissa) versusfrequency of events (ordinate);

FIG. 3 is a scattergram representation for side scatter (SSC onabscissa) versus fluorescence intensity (ordinate);

FIG. 4 is a FACScan™ display for side scatter versus forward scatter;

FIG. 5 is a histogram of fluorescence intensity (abscissa) versusfrequency of events (ordinate);

FIG. 6 is a scattergram representation for side scatter (SSC onabscissa) versus fluorescence intensity (ordinate);

FIG. 7 is a photographic representation of a UV light irradiated agaroseelectrophoresis gel performed on enzyme BAMH nicked PBR322 plasmid DNA;

FIG. 8 is a hybridization saturation plot for equivalents;

FIG. 9 shows hybridization titration curves for fluorescence intensityversus equivalents;

FIG. 10A is a FACScan™ display (SSC vs FSC) of a typical distribution oflysed white cells;

FIG. 10B is a FACScan™ display of the same blood sample as in FIG. 10Awith unstained chicken erytlirocyte nucleii ("CEN") added;

FIG. 10C is a FACScan™ display of the same sample lysed with white bloodcell diluent ("WBC DIL");

FIG. 10D is a FACScan™ display of the same sample as presented in FIGURE10C, but with CEN;

FIG. 10E is a FACScan™ display of the same sample lysed in the samediluent as FIG. 10D, but with 0.5 μg/ml of nucleated red blood cell("NRBC") dye;

FIG. 10F is a FACScan™ display of the same sample lysed in the samediluent as FIG. 10D, but with 0.25 μg/ml of NRBC dye;

FIG. 11 is a graphical representation of the efficiency of 32Pradiolabelled, restriction enzyme-nicked plasmid DNA capture ontophenathridinium activated polystyrene microparticles prepared asdescribed in Example 6;

FIG. 12 is a graphical representation of the efficiency of 32Pradiolabelled plasmid DNA capture onto phenathridinium activatedcarboxymethyl sepharose beads synthesized as described in Example 6;

FIGS. 13A, 13B, and 13C show the structures or representative Imoieties;

FIG. 14 is a reaction scheme for the synthesis of compound 24 and itsprecursors;

FIG. 15 is a schematic representation of intercalator derivatized solidphase microparticle;

FIG. 16 shows the structural formula of compounds 25, 26 and 27;

FIG. 17A, 17B, and 17C show a reaction scheme for the synthesis ofcompounds 28a-42a;

FIG. 18 is a reaction scheme for the synthesis of compound 50 and itsprecursors;

FIG. 19 is a reaction scheme for the synthesis of compounds 55, 55a, andtheir precursors;

FIG. 20 shows the structural formula of compounds 55a-55p;

FIG. 21 is a reaction scheme for the synthesis of compounds 58, 58a, andtheir precursors;

FIG. 22 shows the structural formula of compounds 58a-58p;

FIG. 23 is a reaction scheme for the synthesis of compound 64 and itsprecursors;

FIG. 24 is a reaction scheme for the synthesis of compounds 68, 68a, andtheir precursors;

FIG. 25 shows the structural formula of compounds 68a-68p;

FIG. 26 is a reaction scheme for the synthesis of compounds 71, 71a, andtheir precursors;

FIG. 27 shows the structural formula of compounds 71a-71p;

FIG. 28 is a reaction scheme for the synthesis of compound 80 and itsprecursors;

FIG. 29 is a reaction scheme for the synthesis of compound 85 and itsprecursors;

FIG. 30 is a reaction scheme for the synthesis of compound 90 and itsprecursors;

FIG. 31 is a comparison of relative fluorescence intensities obtainedfrom staining DNA by compound 24, ethidium bromide, propidium iodide,and ethidium homodimer; and

FIG. 32, is a comparison of relative fluorescence intensities obtainedfrom staining DNA by compounds 24, 25, 26, 27, and ethidium bromide.

DETAILED DESCRIPTION

Broadly, the present invention provides an intercalator having an Imoiety bonded to one or more T moiety. The general formula of thecompounds are represented by:

I-(T)_(m)

wherein the I moiety denotes an aromatic or heteroaromatic segment; theT moiety denotes a "tail" or "chain" moiety; and m is an integer from 1to 5, preferably from 1 to 3.

When the I moiety contains a mono-quaternary ammonium functionality, itis accompanied by a monovalent counter anion ("A⁻ "). Examples ofmonovalent counter anion include chloride, bromide, iodide, hydroxide,and hydrogen phosphate.

Improved binding to the DNA molecule of intercalators and substitutedintercalators is achieved which exhibit high affinity for binding,reduced self-quenching, and superior transport kinetics, especially whencompared to ethidium homodimer or other bis-intercalators, by providingintercalator segments with functionalized chains forming compoundshaving the formula: ##STR4## wherein I is an aromatic or heteroaromaticsegment; X is a nitrogen or a sulfur; R, R₁ and R₂ are alkyl, alicyclic,heteroalicyclic, aromatic or heteroaromatic groups; R₃ and R₄ arehydrogens when X is nitrogen or methyl, ethyl or phenyl groups when X isphosphorus or sulfur; k is zero or an integer from 1 to 10; q is zero oran integer from 1 to 10; m is an integer from 1 to 5; n is an integerfrom 2 to 20; o is zero or one; and p is zero or one.

A compound comprised of an intercalator functionalized with chainscontaining heteroatoms and aliphatic, alicyclic, cyclohexyl, aromaticsegments or combinations thereof having the formula: ##STR5## wherein Iis an intercalator segment; X is a main group element phosphorus orsulfur yielding, respectively polyphosphonium or polysulfonium moieties;R, R₁ and R₂ are alkyl, alicyclic, heteroalicyclic, aromatic orheteroaromatic groups; R₃ and R₄ are methyl, ethyl or phenyl groups whenX is phosphorus or sulfur; k is zero or an integer from 1 to 10; q iszero or an integer from 1 to 10; m is an integer from 1 to 5; n is aninteger from 2 to 20; o is zero or one; and p is zero or one; where X issulfur, o or p are zero; and where X is phosphorus, o and p are one.

In another embodiment, it is provided a compound having the generalstructural formula of: ##STR6## wherein I is an aromatic orheteroaromatic segment; X is a nitrogen or a sulfur; R, R₁ and R₂ arethe same or different and are alkyl of one to four carbons, alicyclic offive to six carbons, heteroalicyclic of three to five carbons and one ortwo heteroatom of nitrogen, oxygen or sulfur, aromatic group of benzene,phenyl or naphthyl or heteroaromatic group of one to five carbons andone to four heteroatom of nitrogen, oxygen, or sulfur; R₃ and R₄ arehydrogens when X is nitrogen or methyl, ethyl or phenyl groups when X issulfur; k is zero or an integer from 1 to 10; q is zero or an integerfrom 1 to 10; m is an integer from 1 to 5; n is an integer from 2 to 20;o is zero or one; and p is zero or one.

In yet another aspect, there is provided a compound comprised of anintercalator functionalized with chains containing heteroatoms andaliphatic, alicyclic, cyclohexyl, aromatic segments or combinationsthereof having the formula: ##STR7## wherein I is an aromatic orheteroaromatic segment; X is a main group sulfur yielding polysulfoniummoieties; R, R₁ and R₂ are the same or different and are alkyl of one tofour carbons, alicyclic of five to six carbons, heteroalicyclic of threeto five carbons and one or two heteroatom of nitrogen, oxygen or sulfur,aromatic group of benzene, phenyl or naphthyl or heteroaromatic group ofone to five carbons and one to four heteroatom of nitrogen, oxygen, orsulfur; R₃ and R₄ are hydrogens when X is nitrogen or methyl, ethyl orphenyl groups when X is sulfur; k is zero or an integer from 1 to 10; qis zero or an integer from 1 to 10; m is an integer from 1 to 5; n is aninteger from 2 to 20; o is zero; and p is zero.

A compound comprised of aromatic or heteroaromatic segmentsfunctionalized with positively charged chains having the formula:##STR8## wherein Y is a side chain comprised of positively chargedheteroatoms or metal ions in an aliphatic, alicyclic, heteroalicyclic,aromatic or heteroaromatic group or combinations thereof; I is anaromatic or heteroaromatic segment; X is a heteroatom; R, R₁ and R₂ arealkyl, alicyclic, heteroalicyclic, aromatic or heteroaromatic group; R₃and R₄ are hydrogens when X is nitrogen or methyl, ethyl or phenylgroups when X is phosphorus or sulfur; k is zero or an integer from 1 to10; q is zero or an integer from 1 to 10; m is an integer from 1 to 5; nis an integer from 2 to 20; o is zero or one; p is zero or one; where Xis sulfur, o and p are zero; and where X is phosphorus o and p are one.

A compound as above wherein Y has the formula: ##STR9##

Further, a compound comprised of intercalators functionalized withchains containing metal atoms and alkyl, alicyclic, or aromatic segmentsor combinations having the formula: ##STR10## wherein W is aluminum,boron or a Lewis acid metal; I is an intercalator segment; R, R₁, R₂ andR₃ are alkyl, alicyclic, or aromatic groups; k is zero or an integerfrom 1 to 10; q is zero or an integer from 1 to 10; m is an integer from1 to 5; and n is an integer from 2 to 20.

An intercalator composition functionalized with positively chargedchains where the positive charges are located on an aliphatic,alicyclic, aromatic or the combination thereof with a polyaminic estergroup of main chain polyphosphate, polyphosphonate or polysulfate,having the formula: ##STR11## wherein I is an aromatic or heteroaromaticsegment; P is a phosphorus atom; S is a sulfur atom; Z is an aliphatic,alicyclic or aromatic chain or the combination thereof; n is from 2 to20, preferably 2 or 3; and m is from 1 to 5, preferably, 3-10.

Broadly, the present invention relates to a compound comprised of anintercalator moiety, or a substituted intercalator moiety, derivatizedwith functionalized chains, and the compound has high affinity forbinding to a DNA molecule. In one aspect, the invention relates to useof these intercalator compounds which exhibit improved binding to a DNAmolecule within known methodologies requiring intercalator insertioninto the DNA molecule. Still, the invention relates to enhanced bindingof DNA molecules by an intercalator functioning segment utilized inlabeling, capture, therapeutic insertion, assay and the like, withimproved performance of the intercalator due to the increasedutilization efficiency of the compounds. Improved binding to the DNAmolecule of intercalators and substituted intercalators is achievedwhich exhibit high affinity for binding, reduced self-quenching, andsuperior transport kinetics, especially when compared to ethidiumhomodimer or other bis-intercalators.

The various embodiments of the present invention inclusive of synthesisof the compounds and utilization of said compounds are shown in FIGS.1-9, 10A-10F, 11-30. The information shown in these figures clearlydemonstrates high affinity for binding, reduced self-quenching, andsuperior transport kinetics, especially when compared to ethidiumhomodimer or other bis-intercalators.

FIG. 1 is a FACScan™ display for side scatter versus forward scatter(SSC on abscissa axis and FSC on the ordinate axis) for a typicaldistribution of white cells lysed with WBC DIL diluent with NRBC dyephenathridinium triamine (PTA) 24 and CEN. The quadrant thresholds wereset to preclude the lymphocytes gated on the SSC versus FSC dot plot.

FIG. 2 is a histogram of fluorescence intensity (abscissa) versusfrequency of events (ordinate) for the populations of stained andunstained cells in the presence of phenathridinium triamine (PTA) 24.

FIG. 3 is a scattergram representation for side scatter (SSC onabscissa) versus fluorescence intensity (ordinate) showing theseparation of cells stained with PTA 24 (upper left hand corner, NWquadrant) from unstained cells (remainder) by fluorescence intensity.

FIG. 4 is a FACScan™ display for side scatter versus forward scatter(SSC on abscissa axis and FSC on the ordinate axis) for a typicaldistribution of white cells lysed with WBC DIL diluent with NRBC dyeethidium homodimer and CEN. The quadrant thresholds were set to precludethe lymphocytes gated on the SSC versus FSC dot plot.

FIG. 5 is a histogram of fluorescence intensity (abscissa) versusfrequency of events (ordinate) for the populations of stained andunstained cells in the presence of ethidium homodimer.

FIG. 6 is a scattergram representation for side scatter (SSC onabscissa) versus fluorescence intensity (ordinate) showing theseparation of cells stained with ethidium homodimer (upper left handcorner, NW quadrant) from unstained cells (remainder) by fluorescenceintensity.

FIG. 7 is a photographic representation of a UV light irradiated agaroseelectrophoresis gel performed on BAMH nicked PBR322 plasmid DNA.Loadings of 5 μl of stock solutions were made for lanes 3-14. Thefollowing stock solutions of DNA and intercalator were used to loadlanes 1-14. Lane 1: marker; Lane 2: blank; Lane 3: 20 ng/ml plasmidstained with ethidium bromide; Lane 5: 160 pg/ml plasmid stained withethidium bromide; Lane 6: 40 pg/ml plasmid stained with ethidiumbromide; Lane 7: 20 ng/ml plasmid stained with ethidium homodimer; Lane8: 800 pg/ml plasmid stained with ethidium homodimer; Lane 9: 160 pg/mlplasmid stained with ethidium homodimer; Lane 10: 40 pg/ml plasmidstained with ethidium homodimer; Lane 11: 20 ng/ml plasmid stained withPTA 24; Lane 12: 800 pg/ml plasmid stained with PTA 24; Lane 13: 160pg/ml plasmid stained with PTA 24; Lane 14: 40 pg/ml plasmid stainedwith PTA 24. In all cases, the dye/base pair ratio was 1/20. In allcases, the dye was pre-incubated with the DNA and nopost-gel-electrophoresis staining was done.

FIG. 8 is a hybridization saturation plot for equivalents of d(pT)9added to d(pA)9 at 6.6 micromolar DNA and 3.08 micromolar dye forethidium homodimer and PTA 24 at a dye base pair ratio of 1:4. Graphicalrepresentation of equivalents of complementary oligonucleotide d(pT)9 onthe abscissa added to d(pA)9 versus relative fluorescence intensity onthe ordinate generated by using the protocol described in Example 2 tocompare ethidium homodimer and PTA 24. The concentration of fluorophorein both cases was 3.08 micromolar using a two-fold statisticalcorrection for the 2.0 molar equivalents of phenanthridinium moiety pereach mole of ethidium homodimer. Both curves are normalized tobackground for relative fluorescence. Excitation was at 488 nm (534 nmis the excitation maximum) for this experiment and emission was at 625nm.

FIG. 9 shows hybridization titration curves for fluorescence intensityversus equivalents of d(pT)9 added to d(pA)9 at 6.6 micromolar DNA and3.08 micromolar ethidium bromide and PTA 24. Graphical representation ofequivalents of complementary oligonucleotide d(pT)9 abscissa versusrelative fluorescence intensity (ordinate) generated by using theprotocol described in Example 2 to compare ethidium bromide and PTA 24.The concentration of fluorophore in both cases was 3.08 micromolar. Bothcurves are normalized to background. Excitation was at 488 nm (534 nm ismaximum excitation) and emission was at 625 nm and intensities weremeasured using a Hitachi F-3010 fluorimeter.

FIG. 10A is a FACScan™ display (SSC vs FSC) of a typical distribution ofwhite cells lysed with CD4000 WBC DIL without NRBC dye or CEN. Thequadrant thresholds were set to preclude the lymphocytes gated on theSSC versus FSC dot plot.

FIG. 10B is a FACScan™ display of the same blood sample as in FIG. 10Awith unstained CEN added. As can be seen, the unstained CEN demonstratesome FRL3 auto-fluorescence. The region 1 was set to include all FL3+events in this unstained sample, so that stained cells in the testsamples can be counted in the region 2. The region 3 is set to includethe CEN population only to measure the mean FL3 of the population.

FIG. 10C is a FACScan™ display of the same sample lysed with WBC DILcontaining 1.0 μg/ml of NRBC dye. 1.3% of FL2+ events were detected inμL from the gated lymphocytes and 1.08% of FL3+ events are detected fromthe ungated total white cell population.

FIG. 10D is a FACScan™ display of the same sample as presented in FIG.10C, but with CEN. The region 2 of the FL3 histogram of the ungatedpopulation shows the stained CEN population which has the mean FL3 of3319.8.

FIG. 10E is a FACScan™ display of the same sample lysed in the samediluent as FIG. 10D, but with 0.5 ug/ml of NRBC dye.

FIG. 10F is a FACScan™ display of the same sample lysed in the samediluent as FIG. 10D, but with 0.25 ug/ml of NRBC dye. As can be seen,the stained CEN is still well separated from the white cells.

FIG. 11 is a graphical representation of the efficiency of 32Pradiolabelled plasmid DNA capture onto phenathridinium activatedpolystyrene microparticles synthesized as described in Example 6. A:initial radioactive counts in solution accounting for the total DNAconcentration; B: radioactive counts remaining in solution after removalof DNA via centrifugation as described in Example 6; C: initialradioactive counts on the DNA bound to the microparticle by thephenthridine moiety before release is initiated by NaOH; and D:radioactive counts remaining on the solid after removal of DNA withNaOH.

FIG. 12 is a graphical representation of the efficiency of 32Pradiolabelled plasmid DNA capture onto phenathridinium activatedcarboxymethyl sepharose beads synthesized as described in Example 6. A:initial radioactive counts in solution accounting for the total DNAconcentration; B: radioactive counts remaining in solution after removalof DNA via centrifugation as described in Example 6; C: initialradioactive counts on the DNA bound to the microparticle by thephenthridine moiety before release is initiated by NaOH; and D:radioactive counts remaining on the solid after removal of DNA withNaOH.

The present intercalator compounds are substantially monointercalators,versus the polyfunctional (bis) intercalators. The monointercalatorsaccording to the present invention are most suitable for applicationusing a multitude of intercalators and substituted intercalators whichwhen combined with and functionalized by the various "chains or tails"("T"), where T is the chain comprised of R, R₁, R₂, R₃, R₄, W, X, Y andZ and bounded by the brackets in the previously discussed formulas,which provide high binding to DNA and RNA and lack of self-quenching andsuperior transport kinetics. Representative "I" moieties are given inFIG. 13.

The invention is further defined by the following Examples, whichprovide basis for the FIGURES and are intended to be illustrative, butnot limiting.

EXAMPLE 1

Synthesis of Phenanthridinium Triamine (PTA) 24 and PrecursorIntermediates 20-23

PTA 24, a compound according to the invention, was synthesized throughthe sequence shown in the schematic as shown in FIG. 14. Theexperimental procedures used to obtain product 24 are as illustratedtherein.

Intermediate 21. Starting Intermediate 20, 3,8 Diamino 6-phenylphenathridine (25.0 g, 0.0876 moles), was obtained from the AldrichChemical Company (Milwaukee, Wis.) and added to a single neck 3.0 literround bottom flask under Argon and equipped with a magnetic stir bar anda reflux condenser. To this vessel, 1.0 liter of dry pyridine was addedwhile stirring. Stirring of the resulting suspension was continued for15 minutes until all the solid had dissolved. A catalytic amount ofN,N-dimethylaminopyridine (1.07 g, 0.0086 moles) was added to thissolution while stirring. Acetic anhydride (462 g, 4.9 moles) was thenadded and the resulting reaction mixture was refluxed for 8-12 hours.The reaction mixture was then allowed to cool and the solvent wasremoved in vacuo. For purification of the Product II, a gradient silicagel column was performed using 2.0 liters of 45/40/10/5 Ethylacetate/hexane/ CH₂ Cl₂ CH₃ OH followed by 1.0 liter of 40/40/10/10EtOAc/hexane/ CH₂ Cl₂ /CH₃ OH. Fractions of 10.0 ml were collected andappropriate fractions were recombined and the solvent was removed invacuo. The sticky gum-like residue was then dissolved in hot EtOH (220ml) and precipitated by cooling to 0° C. The mother liquor was decantedoff and 200 ml of fresh EtOH was added. The solid was redissolved byheating and allowed to crystallize at -4° C. for 48 hours. Crystals werecollected from both the mother liquor and the second recrystallizationand were washed with a small amount of cold EtOH and dried under highvacuum for several hours. Isolated yield of pure product after columnchromatography and two recrystallizations was 32%. ¹ H NMR CD₃ OD (300MHz) 9.1 (d, 1H 8.82 Hz), 9.0 (d, 1H, 8.75 Hz), 8.08 (s, 1H), 7.95 (s,1H), 7.92 (d, 1H, 4.5 Hz), 7.8 (m, 3H), 7.65 (m, 3H), 2.45 (s, 6H), 2.35(s, 6H); ¹³ C NMR CD3OD (75.45 MHz) 174.5, 163.7, 145.3, 142.1, 140.6,139.9, 134.4, 133.8, 130.8, 130.6, 129.8, 127.3, 125.9, 125.6, 124.8,27.1, Exact Mass Calc. for C₂₇ H₂₃ N₃ O₄, Calc. 453.1688 exact mass,Obs: 453.1683; CH analysis Calc for C₂₇ H₂₃ N₃ O₄ C: 71.51 H: 5.11 N:9.27 Found C: 71.77 H: 5.10 N:9.20.

Intermediate 22. Intermediate 22 was synthesized from the diamide 21 viaa modification of a literature procedure (Gaugain et al., Biochemistry,Vol. 17, No. 24, 1978, pp. 5071-5078) for quarternization of the diamideof 3,8 Diamino-6-phenyl phenanthridine. Diamide 21 (10.5 g, 0.023 moles)was placed in a 2.0 liter round bottom flask under argon and equippedwith a magnetic stir bar and reflex condenser. 1,3 Dibromopropane (1.0liter, 9.86 moles) was added to this flask and the resultant mixture wasbrought to reflux for 7 hours. The solution was cooled overnight and theprecipitant was filtered and washed with diethyl ether. Obtained 10.44 g(68.7%) of crude material 22. This material was recrystallized from CH₃OH to yield 5.3 g of diacetyl bromide 22. ¹ H NMR CD₃ OD (300 MHz) delta10.75 (s, 1H), 10.45 (s, 1H), 9.3 (s, 1H), 9.09 (d, 9.2 Hz, 1H), 9.04(d, 9.2 Hz, 1H), 8.45 (d, 9.1 Hz, 2.2 Hz, 1H), 8.12 (s, 1H), 8.07 (d 9.0Hz, 1H), 7.95 (m, 3H), 7.85 (m, 3H), 5.0 (t, 9.0 Hz, 3H), 3.6 (t, 6.0Hz, 2H), 2.65, 2.38 7.75 ¹³ C NMR ₂₆ DMSO [????] (75.45 MHz) delta169.9, 169.3, 163.8, 142.1, 139.9, 134.2, 131.5, 130.5, 129.5, 128.4,125.9, 125.4, 123.6, 122.3, 121.6, 119.0, 107.7, 55.7, 30.7, 24.5, 24.2;exact mass Calc. for C₂₆ H₂₉ N₃ O₂ Br₂ free salt (FAB+) 490.1131 Obs:490.1139; CH analysis Calc. for C₂₆ H₂₉ N₃ O₂ Br₂ H: 4.41 C: 54.66 N:7.36 Found H: 4.25 C: 54.65 N: 7.30.

Intermediate 23. Intermediate 22 (5.3 g, 0.0093 moles) was added to a250 ml round bottom flask equipped with a magnetic stir bar and refluxcondenser. Methanol (150 ml) was then added to this flask while stirringunder nitrogen and diethylene triamine (29.2 g, 0.283 moles) was addedwhile stirring was continued. The resultant transparent solution washeated to reflux overnight under nitrogen. This solution was thenallowed to cool to room temperature and poured into distilled H₂ O. Thenthis mixture was concentrated in vacuo until only the H₂ O remained. Anadditional 50-75 ml H₂ O was added and the solution was allowed to coolto 0° C. The solid was then filtered and washed with ice cold water.This material was then redissolved in EtOH and precipitated with 10 NHCl. After filtration of the suspension, the resultant solid wasrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop was also collected from the second filtrate upon standingand by precipitation with EtOH from the first filtrate. These solidswere then combined and the final product 23 (2.5 g) was obtained afterhigh vacuum overnight. ¹ H NMR CD₃ OD (300 MHz) delta 9.2 (s, 1H), 9.05(d, 1H), 8.95 (d, 1H), 8.4 (broad s, 2H), 8.3 (broad s, 1H), 7.9 (broadm, 3H), 7.7 (broad m, 2H), 5.1 (broad m, 1H), 3.9 (broad s, 3H), 3.45(broad m, 2H), 3.85 (broad m, 2H), 2.5 (broad m, 3H), 2.3 (broad m, 3H);MS Calc. for free salt C₃₀ H₃₇ N₆ O₂ (FAB+) 513 Obs: 513.

Product 24. Synthesis and purification of the phenanthidinium triamine,PTA, 24 was accomplished by the following protocol. Triamine 23 (2.35 g,0.0036 moles) was dissolved in 75.0 ml methanol and 75 ml of 4N HCl wasadded. The mixture was refluxed for 2 hours and allowed to cool. Ethanolwas added to this solution and resulting precipitate was filtered andwashed with a minimal amount of cold ethanol. The filtrate wasreconcentrated and fresh ethanol and concentrated aqueous HCl was added.This resulting precipitate was also filtered. Next, this filtrate wasconcentrated to near dryness and Et₂ O was added and the solid filteredoff. The last remaining unfilterable residue was then dissolved inconcentrated HCl and precipitated with EtOH. This material was filteredand washed with ethanol, combined all solid materials from the abovesequence and subjected this material to high vacuum overnight to obtain2.03 g total of the high affinity fluorescent DNA stain PTA 24. ¹ H NMRd₆ -DMSO (300 MHz) delta 10.0 (broad s, 2H), 9.65 (broad s, 2H), 8.68(d, 14.2 Hz, 2H), 8.35 (broad s, 2H), 7.75 (m, 4H), 7.65 (s, 1H) 7.55(d, 9.2 Hz, 2H), 7.35 (d, 9.2 Hz, 2H), 6.28 (s, 1H), 4.5 (broad s, 2H),4.0 (broad s, 8H), 3.4 (broad s, 2H), 3.0 (broad s, 2H), 2.3 (broad m,2H); ¹³ C NMR d₆ -DMSO (75.45 MHz) δ 159.7, 151.1, 134.6, 131.7, 130.9,129.4, 128.8, 128.4, 124.9, 122.9, 120.1, 117.4, 99.6, 51.4, 43.8, 42.5,40.3, 34.9, 18.5; High resolution mass spec. C₂₆ H₃₃ N₆ (FAB+) Calc.429.2767, Obs: 429.2766; CH analysis Calc for C₂₆ H₃₇ N₆ Cl₄.3H₂ O was H6.89 C 49.61 N 13.35 Found H: 6.16; C 49.74 N: 13.08.

EXAMPLE 2

Hybridization Assay

PTA 24 was used to quantitate hybridization when a targetoligonucleotide was titrated with its complementary partner. Acomparison of ethidium bromide staining versus PTA 24 for detecting thishybridization, as per the following protocol, can be found in FIG. 9 andthe comparison of ethidium homodimer versus PTA 24, as per the followingprotocol, can be found in FIG. 8. Complementary strands of DNA(oligodeoxythymidylic acid, d(pT)₉ and oligodeoxyadenylic acid (d(pA)₉)were obtained from the Sigma Chemical Co. in St. Louis, Mo. A stocksolution of d(pA)9 was made at 5 units/ml of 0.05M TRIS, 0.2N NaCl, 1MMEDTA, pH 8.4. For polyA, ε=8.4 AU/mM cm or 8,400 M⁻¹ cm⁻¹ ; therefore,with 9 base pairs for d(pA)9, the ε is 75,600 M⁻¹ cm⁻¹. This stock wasthen diluted 10× to obtain stock at 6.61×10⁻⁶ M, or 6.6 μM. The d(pT)₉stock was made at 25 units/5.0 ml and used for titration without furtherdilution in the same buffer. Since the ε for polyT is 8.15 AU/mM cm or8,150 M-1cm-1 per base pair, or 73,350 M-1cm-1per oligo, theconcentration of the oligo stock was 68 μM in DNA molecules. A titrationwas performed using a Hitachi F4010 Fluorescence Spectrophotometerequipped with 0.5 ml microcells to obtain fully corrected spectra and anexcitation wavelength of 488-550 nm (optimal around 534) and an emissionwavelength of 600-650 nm (optimal around 625). Equivalents of d(pT),were added at the following increments: 0.02, 0.05, 0.080, 0.150, 0.300,0.500, 0.700, 1.00, 2.00, 5.00 equivalents. Each sample in the titrationcurve was prepared individually by dividing the initial d(pA)9 stockinto 10×1.0 ml aliquots. The addition of complement was thenaccomplished by micropipetting an appropriate amount (2, 5, 8, 15, 30,50, 70, 100, 200, and 500 μl, respectively) of d(pT)9 stock to each of aseries of the 10 aliquots. Each aliquot, obtaining progressively largermolar ratios of the two complementary strands, was incubated at ambienttemperature for 15 minutes, the dye was added as 20.0 μl aliquots of a154 μM solution of the dye in 0.05M TRIS, 0.2N NaCl, 1 mM EDTA, pH 8.4buffer. This corresponds to a dye/DNA b.p. ratio of 1/20 at saturationwith complementary oligo. Overall concentrations of dye and oligo varyin the saturation plot because of the use of varied increments additionsfrom the same stock solution. After an additional 15 minute incubationtime, the relative fluorescence intensity was then read at 625 nm andrecorded to generate a standard curve which is directly proportional tothe quantity of dsDNA hybridization, or target sequence, under theseconditions. The background fluorescence, or initial residualfluorescence, is then subtracted out as a constant for all curves forcomparison of the various titration curves on the same graph.

EXAMPLE 3

Gel Electrophoresis Application

An agarose gel was run to compare the staining intensity of ethidiumbromide (Aldrich Chemical Co., Milwaukee, Wis.), ethidium homodimer -1(Molecular Probes, Cat. # E 1169, Eugene, Oreg.), and PTA 24 stain.Plasmid, pBR322, at 2.1 mg in 7 ml stock was incubated at 37° C. for 1hour with 1 ml of BAMH restriction enzyme with 2 ml 10× React2 Bufferand diluted to 20 ml total with 10 ml H₂ O. This mixture was then usedto prepare 3 stocks of nicked pBR322 plasmid at 0.63 mg per 6 ml foreach vial. Each of these stocks were diluted further with H₂ O and 20%glycerol to final DNA stocks of 20 ng/ml, 800 pg/ml, 160 pg/ml, and 40pg/ml with a 1:4 ratio of dye to DNA base pairs in each for a total of12 stocks. A 5 ml aliquot of each stock was loaded into 12 separatelanes in agarose gel and electrophoresis was run for 30 minutes in 4 mMTRIS, pH8.2, with 0.01 mM EDTA buffer. The gel was then removed andphotographed under exposure to U.V. light in a conventional gel box.

EXAMPLE 4

Protocol for Synthesis of Intercalator Activated Carboxymethyl StyreneMicroparticle Capture Reagent

The synthesis of intercalator derivatized solid phase microparticle (MP)capture reagent was accomplished by the scheme depicted in the followingschematic and effected by the following procedure:

A 45 aliquot of 0.275±μm microparticles (Seradyne, Indianapolis, Ind.)were placed in a 4 ml vial and the surfactant was exchanged out usingBio-Rex 501-D ion exchange mixed bed resin (Bio-Rad, Richmond, Calif.).After gentle shaking for 2 hours, the resin was filtered out from themixture by using a coarse fritted glass funnel equipped with a reducedpressure collection chamber. The sample was diluted to a concentrationof MP at 10% solids by weight. The total amount of equivalents ofreactive carboxylic acid were calculated from the titrationspecifications of the vendor.

A stock solution of sulfo N-hydryoxysuccinimide (Pierce, Rockford, Ill.)was made at 11 mg/ml (20 mM) in H₂ O and a stock solution of EDAC (SigmaChemical Co., St. Louis, Mo.) at 10 mg/ml (5 mM) was made in H₂ O. Fiveequivalents of EDAC (290 μl stock) was added to the carboxymicroparticlereaction mixture, followed by 5.0 equivalents of sulfoN-hydryoxysuccinimide (330 μl stock). This mixture was allowed toincubate at room temperature for 2 hours and then a 2.0 molar equivalentof PTA 24 (4 mg) was added at a concentration of 8 mg/400 μl, or 2.0mg/100 μl in pH 8.0 0.1 N NaCl 0.1N Pi phosphate buffer.N-hydryoxysuccinimide (Pierce) can be substituted for sulfoN-hydryoxysuccinimide if it is first dissolved in a stock of DMF(Dimethyl formamide) and aliquoted as described above. After allowing 24hours for complete reaction, the free dye was then removed bycentrifugation, removal of mother liquor, and resuspension for severalattempts until the solution went clear and no more dye was extractedfrom the samples. The purified capture reagent was then diluted to astock of 2-4% solids in H₂ O.

A general schematic representation of this Example is given in FIG. 15.

EXAMPLE 5

Solid Phase DNA Capture

CM (carboxy modified) Sepharose was obtained from Sigma Chemical Co.(St. Louis, Mo.) in an ethanol/H₂ O mixture. The solution was estimatedat 50% solids based on total volume occupied by the solid and liquidportions on extended standing. This suspension was then mixed uniformlyand diluted to 10% solids. 200 μl of this stock was removed andcalculated at 0.12 meq/gram to be 0.012 meq of acid total. Stock of EDACand N-hydryoxysuccinimide were prepared and 5.0 equivalents of eachactivating reagent were added to this suspension. For this preparation,13.2 mg (in 1.32 ml) HOSuc and 11.25 mg EDAC (in 1.02 ml) were used and8.0 mg total of the PTA 24 intercalator. After incubation for 2-24hours, the suspensions were then cleaned by repeated washing and gentlecentrifugation steps until no more color was removed from the solid upondilution. A stock was prepared at 10% solids in H₂ O. Note that controlswere run with PTA 24 modified and non-modified solid phases and minimalnon-specific capture of DNA occurred with the underivatized materials.

EXAMPLE 6

Protocol for DNA Capture by Intercalator Modified Solid Phase

1. Place 50 μl of activated microparticles in a 1.5 ml eppendorf.

2. Add 150 μl of PBS and 1-20 u of a 5.0 kb linearized plasmidend-labeled with ³² P. Alternatively, added 1-50 μl of biological sampleor another purified DNA.

3. Mix by rotation for one hour at ambient temperature.

4. Pellet the microparticles by centrifugation at 5,000 rpm for 5minutes.

5. Wash one or two times with 200 μl of PBS.

6. Release the DNA by adding 50μ of 0.5 M NaOH and mix for 15 minutes atambient temperature.

7. Centrifuged and collected the supernatant, which contains releasedDNA.

The efficiency of DNA capture was measured using ³² P radiolabelledplasmid DNA in the above-described protocol. The results are found inFIG. 11 using the intercalator modified polystyrene microparticlesprepared as per Example 4 and FIG. 12 and using the intercalatormodified CM Sepharose beads prepared per Example 5. The data indicatesthat the DNA binding to the intercalator modified solid phase wasspecific and induced by the covalent attachment of intercalator 24 tothe solid phase.

EXAMPLE 7

Relative Staining Intensities of Ethidium Homodimer and PhenanthridiniumTriamine 24 in a Flow Cytometric Study of Chicken Erythrocyte Nuclei(CEN)

Protocol: 50 μl of whole blood sample from two in-house donors and 3 μlof CEN suspension was added to 1.0 ml of pre-warmed at 40° C. WBC DILwithout and with the NRBC dye at 1 μg/ml concentration, mixed,introduced to the FACScan™ and 20" readings were acquired. Chickenerythrocyte nuclei (CEN) were used to measure the brightness of the FL3staining (mean FL3 of CEN). The whole blood samples used were about 4-5hours old. The data for this experiment is shown in FIGS. 1-6.

EXAMPLE 8

Comparative Performance of Reduced Phenathidinium Triamine 24 DyeConcentrations Relative to Ethidium Homodimer

The effect of a reduction in PTA 24 dye concentration (FIGS. 10A-F)relative to ethidium homodimer was demonstrated as follows:

Method: The experiment was designed to show the correlation between thedye concentration and the percent of FL2+ events in the UL quadrant onthe FL1 versus FL2 dot plots. The WBC DIL used contained 0.5%weight/volume of ammonium chloride, 0.075% of volume of formaldehyde,0.01% weight/volume of saponin, 0.01% weight/volume of potassiumbicarbonate, and 20 mM acetate buffer with a pH of about 6.0 and anosmolality of about 270 mOsm per liter. 50 μl of whole blood sample fromeach of two in-house donors was added to 1.0 ml of pre-warmed at 40° C.WBC DIL without and with the NRBC dye of varying concentration (0.25,0.50, 0.75, and 1.0 μg/ml), mixed, introduced to the FACScan™ and 20"readings were acquired. Chicken erythrocyte nuclei (CEN) suspension wasused to measure the brightness of the FL3 staining (mean FL3 of CEN).The whole blood samples used for this experiment were about 6 hours old.

It was observed that the CEN DNA is essentially indistinguishable fromthe background without the PTA 24 (FIG. 10B) and that the dyeconcentration can be reduced to 75% of that of ethidium homodimer (FIG.5) and still maintain acceptable separation from the background (FIG.10F). Such a reduction can lead to significantly reduced non-specificbinding and substantial savings in dye usage.

EXAMPLE 9

Viability Dyes on the Coulter Elites Flow Cytometer

Cell Isolation Protocol: Each tube of ficol isolated cells were treatedas follows: PBS with 0.1% NaAzide and 1.0% albumin (Sigma catalogue#1000-3) Ficol specific gravity 1.119 (Sigma Histopague catalogue#1119-1).

10 ml of whole blood (EDTA anticoagulant) was diluted with 10 ml ofPBSW. Into 4, 15 ml conical bottom tubes, 5 ml of the diluted blood waslayered over 5 ml of ficol. The tubes were spun for 30 minutes at 400×G.The interface layer which contains the lymphocytes, monocytes,granulocytes and platelets was aspirated and washed once in 5 ml PBS, bycentrifuging tubes at 300×G for 6 minutes. The cell pellet wasresuspended in PBS, cells counted, and adjusted to 8.5×106 cells per ml.

Cell Staining Protocol:

Dye Solutions:

PTA 24--Stock solution 10 ug/ml made by dissolving PTA 24 in PBS with0.1% NaAzide.

Propidium iodide (P.I.)--Stock solution 0.5 mg/ml made by dissolvingP.I. in PBS with 0.1% NaAzide.

P.I. Staining:

In 12×75 mm tube, 117.6 μl of cells was mixed gently with 14.7 μl ofP.I. stock dye solution. After 20 seconds, the tube was placed on aCoulter Elite™ flow cytometer and data collected.

Procedure from "Discrimination of Viable and Non-Viable Cells UsingProdidium Iodide in Two Color Immunofluorescence", Cytometry by Sasakiet al., Vol. 8, 1987, pp. 413-420.

PTA 24 Staining:

In 12×75 mm tube, 23.5 μl of cells was gently mixed with 76 μl of PTA 24stock dye solution. After 20 seconds, the tube was placed on a CoulterElite™ flow cytometer and data collected.

Trypan Blue Staining:

In 12×75 mm tube, 5 μl of working solution Trypan Blue and 5 μl of cellswere gently mixed and cells counted in a mehacytometer using standardwhite light illumination. A minimum of 500 cells were counted within 3minutes of staining.

Procedure from Selected Methods in Immunology by Mishell and Shiigi,1980, pp. 16-17.

Flow cvtometer protocol: Cells analyzed on the Elite™ flow cytometer(Coulter Electronics, Inc.).

Samples were excited with an argon laser at 488 nm and 15 mW of power.Data was gated on the basis of size and granularity to exclude red bloodcells, platelets and debris. The linear dye fluorescence of the gateddistribution was analyzed using unstained cells as a control. Thepercent positive events (dead cells) and the mean fluorescence of thedead cell distribution were recorded.

                  TABLE 1                                                         ______________________________________                                        Viability Dyes on the Coulter Elite ™ Cytometer                            Time Point  Sample    % Positive (Dead Cells)                                 ______________________________________                                         5 hr       P.I.      2.5                                                                                                 2.2                                                                    1.4                                       27 hr                                     6.3                                                                            7.7                                                                   4.8e                                      103 hr                                     26.8                                                                           19.1                                                                   10.2                                     ______________________________________                                    

EXAMPLE 10

Synthesis of Compound 25

Compound 22 (0.075 g, 0.00013 moles) was added to a 50 ml round bottomflask equipped with a magnetic stir bar and reflux condensor. Methanol(10 ml) was then added to this flask while stirring under nitrogen andtris(2-aminoethyl)amine (1.016 g, 0.00518 moles) was added whilestirring was continued. The resultant transparent solution was heated toreflux overnight under nitrogen. This solution was then allowed to coolto room temperature and poured into distilled H₂ O. Then this mixturewas concentrated in vacuo until only the H₂ O remained. An additional50-75 ml H₂ O was added and the solution was allowed to cool to 0° C.The solid was then filtered and washed with ice cold water. Thismaterial was then redissolved in EtOH and precipitated with 10 N HCl.After filtration of the suspension, the resultant solid wasrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop was also collected from the second filtrate upon standingand by precipitation with EtOH from the first filtrate. These solidswere then combined and the final product 23 (2.5 g) was obtained afterhigh vacuum overnight. Material was then carried through to the nextstep of hydrolysis.

The amine residue from above was dissolved in 10.0 ml methanol and 15 mlof 4N HCl was added. The mixture was refluxed for 2 hours and allowed tocool. Ethanol was added to this solution and resulting precipitate wasfiltered and washed with a minimal amount of cold ethanol. The filtratewas reconcentrated and fresh ethanol and concentrated aqueous HCl wasadded. This resulting precipitate was also filtered. Next, this filtratewas concentrated to near dryness and Et₂ O was added and the solidfiltered off. The last remaining unfilterable residue was then dissolvedin concentrated HCl and precipitated with EtOH. This material wasfiltered and washed with ethanol, combined all solid materials from theabove sequence and subjected this material to high vacuum overnight toobtain the of the high affinity fluorescent DNA stain phenathridiumamine derivative 25. High resolution mass spec. C₂₈ H₃₈ N₇ (FAB+) Calc.472.3189 Obs: 472.3191.

The structural formula of compound 25 is shown in FIG. 16.

EXAMPLE 11

Synthesis of Compound 26

Intermediate 22 (0.2 g, 0.406 mmoles) was added to a 50 ml round bottomflask equipped with a magnetic stir bar and reflux condenser. Methanol(10 ml) was then added to this flask while stirring under nitrogen and1,4 Bisamino(3-aminopropyl)piperazine (3.3 g, 16.26 mmoles) was addedwhile stirring was continued. The resultant transparent solution washeated to reflux overnight under nitrogen. This solution was thenallowed to cool to room temperature and poured into distilled H₂ O. Thenthis mixture was concentrated in vacuo until only the H₂ O remained. Anadditional 50-75 ml H₂ O was added and the solution was allowed to coolto 0° C. The solid was then filtered and washed with ice cold water.This material was then redissolved in EtOH and precipitated with 10 NHCl. After filtration of the suspension, the resultant solid wasrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop was also collected from the second filtrate upon standingand by precipitation with EtOH from the first filtrate. These solidswere then combined and the diamine amine product was obtained after highvacuum overnight. Material was then carried through to the next step ofhydrolysis.

The amine residue from above was dissolved in 10.0 ml methanol and 15 mlof 4N HCl was added. The mixture was refluxed for 2 hours and allowed tocool. Ethanol was added to this solution and resulting precipitate wasfiltered and washed with a minimal amount of cold ethanol. The filtratewas reconcentrated and fresh ethanol and concentrated aqueous HCl wasadded. This resulting precipitate was also filtered. Next, this filtratewas concentrated to near dryness and Et₂ O was added and the solidfiltered off. The last remaining unfilterable residue was then dissolvedin concentrated HCl and precipitated with EtOH. This material wasfiltered and washed with ethanol, combined all solid materials from theabove sequence and subjected this material to high vacuum overnight toobtain the of the high affinity fluorescent DNA stain phenathridiumamine derivative 26. Mass Spec. C₂₆ H₃₀ N₅ (FAB+) Calc. 412, Obs: 412.

The structural formula of compound 26 is shown in FIG. 16.

EXAMPLE 12

Synthesis of Compound 27

Intermediate 22 (0.06 g, 0.12 mmoles) was added to a 50 ml round bottomflask equipped with a magnetic stir bar and reflux condensor. Methanol(10 ml) was then added to this flask while stirring under nitrogen andpiperazine-1-carboxyaldehyde (1.37 g, 12.0 mmoles) was added whilestirring was continued. The resultant transparent solution was heated toreflux overnight under nitrogen. This solution was then allowed to coolto room temperature and poured into distilled H₂ O. Then this mixturewas concentrated in vacuo until only the H₂ O remained. An additional50-75 ml H₂ O was added and the solution was allowed to cool to 0° C.The solid was then filtered and washed with ice cold water. Thismaterial was then redissolved in EtOH and precipitated with 10 N HCl.After filtration of the suspension, the resultant solid wasrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop was also collected from the second filtrate upon standingand by precipitation with EtOH from the first filtrate. These solidswere then combined and the diamine amine product was obtained after highvacuum overnight. Material was then carried through to the next step ofhydrolysis.

The amine residue from above was dissolved in 10.0 ml methanol and 15 mlof 4N HCl was added. The mixture was refluxed for 2 hours and allowed tocool. Ethanol was added to this solution and resulting precipitate wasfiltered and washed with a minimal amount of cold ethanol. The filtratewas reconcentrated and fresh ethanol and concentrated aqueous HCl wasadded. This resulting precipitate was also filtered. Next, this filtratewas concentrated to near dryness and Et₂ O was added and the solidfiltered off. The last remaining unfilterable residue was then dissolvedin concentrated HCl and precipitated with EtOH. This material wasfiltered and washed with ethanol, combined all solid materials from theabove sequence and subjected this material to high vacuum overnight toobtain the of the high affinity fluorescent DNA stain phenathridiumamine derivative 27. Mass Spec. C₃₂ H₄₄ N₇ (FAB+) Calc. 526, Obs: 526.

The structural formula of compound 27 is shown in FIG. 16.

EXAMPLE 13

Synthesis Of Compounds 28a-42a and their Related Compounds

The reaction scheme for the general synthesis of compounds 28a-42a, andtheir respective precursors, is given in FIG. 17.

Generally, compound 22 (0.0081 moles) is added to a 250 ml round bottomflask equipped with a magnetic stir bar and reflux condenser. Methanol(150 ml) is then added to this flask while stirring under nitrogen andan appropriate amine (0.283 moles) is added while stirring is continued.The resultant solution is heated to reflux overnight under nitrogen.This solution is then allowed to cool to room temperature and is pouredonto distilled H₂ O. Then, this mixture is concentrated in vacuo untilonly the H₂ O remains. An additional 50-75 ml H₂ O is added and thereaction mixture is cooled to 0° C. The solid is filtered and washedwith ice cold water. This material is then redissolved in EtOH andprecipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product, depending on theamine used to carry out the reaction, is obtained and is subjected tohigh vacuum overnight. Characterization can be effected by calculatingthe molecular mass of the free base amine from the exact isotopic massformulas well known to those skilled in the art and comparing theresultant mass with that obtained by a high resolution mass spectrometrymolecular weight determination such as are well known to those skilledin the art.

Listed below are the products that can be obtained from theircorresponding starting amines.

4-amino-1-benzylpiperidine to yield compound 28a

spermine to yield compound 29a

pyridine to yield compound 30a

2-(2-Aminoethyl)-1-methylpyrrolidine to yield compound 31a

1-(2-Aminoethyl)pyrrolidine to yield compound 32a

1-(2-Aminoethyl)piperidine to yield compound 33a

2-(2-Aminoethyl)pyridine to yield compound 34a

1-(2-Aminoethyl)piperazine to yield compound 35a

4-(2-Aminoethyl)morpholine to yield compound 36a

1-Amino4-(2-hydroxyethyl)piperazine to yield 37a

4-(Aminomethyl)piperidine to yield 38a

2-(Aminomethyl)pyridine to yield 39a

aniline to yield 40a

1-(3-Aminopropyl)imidazole to yield 41a

4-(3-Aminopropyl)morpholine to yield 42a

Synthesis and purification of the final products 28b-42b, depending onthe starting amide used, 28a-42a, can be accomplished by the followinggeneral procedure. The appropriate amide (0.0036 moles) is dissolved in75.0 ml methanol and 75 ml of 4N HCl is added. The mixture is refluxedfor 2 hours and allowed to cool. Ethanol is added to this solution andresulting precipitate is filtered and washed with a minimal amount ofcold ethanol. The filtrate is reconcentrated and fresh ethanol andconcentrated aqueous HCl is added. This resulting precipitate is alsofiltered. Next, this filtrate is concentrated to near dryness and Et₂ Ois added and the solid filtered off. The last remaining unfilterableresidue is then dissolved in concentrated HCl and precipitated withEtOH. This material is filtered and washed with Ethanol. All solidmaterials are combined from the above sequence and is subjected to highvacuum overnight to obtain the high affinity fluorescent DNA stain1b-15b, depending on the amine used as described above. Characterizationcan be effected by calculating the molecular mass of the free base aminefrom the exact isotopic mass formulas well known to those skilled in theart and comparing the resultant mass with that obtained by a highresolution mass spectrometry molecular weight determination such as arewell known to those skilled in the art.

Listed below are the products that can be obtained from theircorresponding starting amides.

amide 28a to yield product 28b

amide 29a to yield product 29b

amide 30a to yield product 30b

amide 31a to yield product 31b

amide 32a to yield product 32b

amide 33a to yield product 33b

amide 34a to yield product 34b

amide 35a to yield product 35b

amide 36a to yield product 36b

amide 37a to yield product 37b

amide 38a to yield product 38b

amide 39a to yield product 39b

amide 40a to yield product 40b

amide 41a to yield product 41b

amide 42a to yield product 42b

EXAMPLE 14

Synthesis of Compound 50 and its Related Compounds

The reaction scheme for the general synthesis of compound 50 and itsprecursors is given in FIG. 18.

2-Methylbenzathiazole 43, from the Aldrich Chemical Company (Milwaukee,Wis.), is alkylated to produce compound 44 using methyl iodide byadapting procedures such as found in P. L. Southwick and A. S. Waggoneret al., U.S. Pat. No. 4,981,977, Jan. 1, 1991, or in Ernst et al.,Cytometry, 10, 1989, pp. 3-10. Compound 45 is obtained from the AldrichChemical Company and is reacted with 3-bromo-1-propanol (also availablefrom the Aldrich Chemical Company) adapting procedures of Gaugain, et.al, Biochemistry, Vol. 17, No. 24, 1978, pp. 5071-5078. The condensationof compound 44 and 46 is effected by adapting procedures found in Hamer,Francis, "Heterocyclic Compounds, Cyanine Dyes and Related Compounds",Wiley, 1964, pg. 37 to yield compound 47. Compound 48 can then beobtained by converting the alcohol to the tosylate by using proceduressuch as found in Wiberg, K. et al., J. Am. Chem. Soc., 92 (3), 1970, pp.553-564. The tosylate 48 is then converted to the bromide via anucleophilic displacement reaction with sodium bromide as per Wilt, J.,J. Org. Chem., 35 (8), 1970, pp.2803-2806 to yield compound 49.Alternatively, a one step procedure is provided in Hooz, J. et al., Can.J. Chem., 46, 1968, pp. 86-87. Compound 49 can then be reacted withdiethylene triamine (available from the Aldrich Chemical Company) as perthe following procedure.

Compound 49 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and thediethylene triamine (0.283 moles), which is available from the AldrichChemical Company is added while stirring is continued. The resultantsolution is heated to reflux overnight under nitrogen. This solution isthen allowed to cool to room temperature and is poured onto distilled H₂O. Then, this mixture is concentrated in vacuo until only the H₂ Oremains. An additional 50-75 ml H₂ O is added and the reaction mixtureis cooled to 0° C. The solid is filtered and washed with ice cold water.This material is then redissolved in EtOH and precipitated with 10 NHCl. After filtration of this suspension, the resultant solid isrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop is also collected from the second filtrate upon standing andby precipitation with EtOH from the first filtrate. These solids arethen combined and the final product 50 is obtained and is subjected tohigh vacuum overnight. Characterization can be effected by calculatingthe molecular mass of the free base amine from the exact isotopic massformulas well known to those skilled in the art and comparing theresultant mass with that obtained by a high resolution mass spectrometrymolecular weight determination such as are well known to those skilledin the art.

Amine derivatives of compound 49 can be synthesized as follows. Compound49 (0.0081 moles) is added to a 250 ml round bottom flask equipped witha magnetic stir bar and reflux condenser. Methanol (150 ml) is thenadded to this flask while stirring under nitrogen and the appropriateamine selected from the following list (0.283 moles), which areavailable from the Aldrich Chemical Company, is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product 49a-49o can beobtained and is subjected to high vacuum overnight.

Listed below are the products that can be obtained from theircorresponding amines.

4-amino-1-benzylpiperidine to yield 49a

spermine to yield to yield 49b

pyridine to yield to yield 49c

2-(2-Aminoethyl)-1-methylpyrrolidine to yield 49d

1-(2-Aminoethyl)pyrrolidine to yield 49e

1-(2-Aminoethyl)piperidine to yield 49f

2-(2-Aminoethyl)pyridine to yield 49g

1-(2-Aminoethyl)piperazine to yield 49h

4-(2-Aminoethyl)morpholine to yield 49i

1-Amino4-(2-hydroxyethyl)piperazine to yield 49j

4-(Aminomethyl)piperidine to yield 49k

2-(Aminomethyl)pyridine to yield 49l

aniline to yield 49m

1-(3-Aminopropyl)imidazole to yield 49n

4-(3-Aminopropyl)morpholine to yield 49o

Characterization can be effected by calculating the molecular mass ofthe free base amine from the exact isotopic mass formulas well known tothose skilled in the art and comparing the resultant mass with thatobtained by a high resolution mass spectrometry molecular weightdetermination such as are well known to those skilled in the art.

Since the structures of compounds 49a-49o are unambiguous from the abovegeneric procedures, there structures are not given.

EXAMPLE 15

Synthesis of Compounds 55 and 55a-55b

The reaction scheme for the general synthesis of compounds 55, 55a, andtheir precursors, is given in FIG. 19. The structural formula ofcompounds 55a-55p are given in FIG. 20.

2-Methylbenzathiazole, 43, is obtained from the Aldrich Chemical Company(Milwaukee, Wis.). It is alkylated to produce compound 51 using3-bromol-propanol adapting procedures such as found in Gaugain et al.,Biochemistry, 17 (24), 1978, 5071-5078. Compound 45 is obtained from theAldrich Chemical Company and is reacted with 3-bromo-1-propanol (alsoavailable from the Aldrich Chemical Company) by also adapting proceduressuch as found in Gaugain et al., Biochemistry 17 (24) 1978, 5071-5078.The condensation of compound 51 and 52 is effected by adaptingprocedures found in Hamer, Francis, "Heterocyclic Compounds, CyanineDyes and Related Compounds", Wiley, 1964, pg. 37 to yield compound 52.Compound 53 is then obtained by converting the alcohol to the tosylateby using procedures such as found in Wiberg, K. et al., J. Am. Chem.Soc., 92 (3), 1970, pp. 553-564. The tosylate 53 is then converted tothe bromide via a nucleophilic displacement reaction with sodium bromideas per Wilt, J., J. Org. Chem., 35 (8), 1970, pp. 2803-2806 to yieldcompound 54.

Alternatively, a one step procedure is provided in Hooz, J. et al., Can.J. Chem., 46, 1968, pp. 86-87.

Compound 54 can then be reacted with diethylene triamine or otherappropriate amine from the following list (available from the AldrichChemical company) to yield compounds 54a-54p in accordance with thefollowing procedure.

Compound 54 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to the flask while stirring under nitrogen and thediethylene triamine or other appropriate base (0.283 moles), availablefrom the Aldrich Chemical Company, is added while stirring is continued.The resultant solution is heated to reflux overnight under nitrogen.This solution is then allowed to cool to room temperature and is pouredonto distilled H₂ O. Then, this mixture is concentrated in vacuo untilonly the H₂ O remains. An additional 50-75 ml H₂ O is added and thereaction mixture is cooled to 0° C. The solid is filtered and washedwith ice cold water. This material is then redissolved in EtOH andprecipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product 9a-p is obtainedand is subjected to high vacuum overnight. Characterization can beeffected by calculating the molecular mass of the free base amine fromthe exact isotopic mass formulas well known to those skilled in the artand comparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

As long as the ratios of reagents and solvents are held constant, onecan scale up or down the amounts of reagents using the method of ratioand proportions well known to those skilled in the art. Listed below arethe products that can be obtained from their corresponding startingamines.

diethylene triamine to yield compound 54a

4-amino-1-benzylpiperidine to yield compound 54b

spermine to yield compound 54c

pyridine to yield compound 54d

2-(2-Aminoethyl)-1-methylpyrrolidine to yield compound 54e

1-(2-Aminoethyl)pyrrolidine to yield compound 54f

1-(2-Aminoethyl)piperidine to yield compound 54g

2-(2-Aminoethyl)pyridine to yield compound 54h

1-(2-Aminoethyl)piperazine to yield compound 54i

4-(2-Aminoethyl)morpholine to yield compound 54j

1-Amino4-(2-hydroxyethyl)piperazine to yield 54k

4-(Aminomethyl)piperidine to yield 54l

2-(Aminomethyl)pyridine to yield 54m

aniline to yield 54n

1-(3-Aminopropyl)imidazole to yield 54o

4-(3-Aminopropyl)morpholine to yield 54p

EXAMPLE 16

Synthesis of Compounds 58 and 58a-58p

The reaction scheme for the general synthesis of compounds 58, 58a andtheir precursors, is given in FIG. 21. The structural formula ofcompounds 58a-58p are given in FIG. 22.

2-Methylbenzathiazole 43 is obtained from the Aldrich Chemical Company(Milwaukee, Wis.). It is alkylated to produce compound 51 using3-bromo-1-propanol by adapting procedures Gaugain, et. al, Biochemistry,Vol. 17, No. 24, 1978, pp. 5071-5078. Compound 45 is obtained from theAldrich Chemical Company and is reacted with methyl iodide (alsoavailable from the Aldrich Chemical Company) to yield compound 55 usingprocedures such as found in Southwick et al. U.S. Pat. No. 4,981,977,Jan. 1, 1991, or in Ernst et al., Cytometry, 10, 1989, pp. 3-10. Thecondensation of compound 51 and 55 is effected by adapting proceduresfound in Hamer, Francis, "Heterocyclic Compounds, Cyanine Dyes andRelated Compounds", Wiley, 1964, pg. 37 to yield compound 56. Compound57 is then obtained by converting the alcohol to the tosylate by usingprocedures such as found in Wiberg, K. et al., J. Am. Chem. Soc., 92(3), 1970, pp. 553-564. The tosylate 57 is then converted to the bromidevia a nucleophilic displacement reaction with sodium bromide describedby Wilt, J., J. Org. Chem., 35 (8), 1970, pp. 2803-2806 to yieldcompound 58.

Alternatively, a one step procedure can be used as provided in Hooz, J.et al., Can. J. Chem., 46, 1968, pp. 86-87.

Compound 58 can then be reacted with diethylene triamine or otherappropriate amine (available from the Aldrich Chemical company) asfollows.

Compound 58 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and thediethylene triamine or other appropriate amine (0.283 moles), which isavailable from the Aldrich Chemical Company is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product 58a-58p is obtainedand is subjected to high vacuum overnight. Characterization can beeffected by calculating the molecular mass of the free base amine fromthe exact isotopic mass formulas well known to those skilled in the artand comparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

As long as the ratios of reagents and solvents are held constant, onecan scale up or down the amounts of reagents using the method of ratioand proportions well known to those skilled in the art.

Listed below are the products that can be obtained from theircorresponding amines.

diethylene triamine to yield compound 58a

4-amino-1-benzylpiperidine to yield compound 58b

spermine to yield compound 58c

pyridine to yield compound 58d

2-(2-Aminoethyl)-1-methylpyrrolidine to yield compound 58e

1-(2-Aminoethyl)pyrrolidine to yield compound 58f

1-(2-Aminoethyl)piperidine to yield compound 58g

2-(2-Aminoethyl)pyridine to yield compound 58h

1-(2-Aminoethyl)piperazine to yield compound 58i

4-(2-Aminoethyl)morpholine to yield compound 58j

1-Amino4-(2-hydroxyethyl)piperazine to yield 58k

4-(Aminomethyl)piperidine to yield 58l

2-(Aminomethyl)pyridine to yield 58m

aniline to yield 58n

1-(3-Aminopropyl)imidazole to yield 58o

4-(3-Aminopropyl)morpholine to yield 58p

EXAMPLE 17

Synthesis of Compound 64 and its Related Compounds

The reaction scheme for the general synthesis of compound 64 and itsprecursors is given in FIG. 23.

2-Methylbenzoxazole, 59, is obtained from the Aldrich Chemical Company(Milwaukee, Wis.). It is alkylated to produce compound 60 using methyliodide by adapting procedures such as found in Southwick et al. U.S.Pat. No. 4,981,977, Jan. 1, 1991 or in Ernst et al., Cytometry, 10,1989, pp. 3-10. Compound 45 is obtained from the Aldrich ChemicalCompany and is reacted with 3-bromo-1-propanol (also available from theAldrich Chemical Company) by adapting procedures Gaugain, et. al,Biochemistry, 5 Vol. 17, No. 24, 1978, pp. 5071-5078. The condensationof compound 60 and 46 is effected by adapting procedures found in Hamer,Francis, "Heterocyclic Compounds, Cyanine Dyes and Related Compounds",Wiley, 1964, pg. 37 to yield compound 61. Compound 62 can then beobtained by converting the alcohol to the tosylate by using proceduressuch as found in Wiberg, K. et al., J. Am. Chem. Soc., 92 (3), 1970, pp.553-564. The tosylate 62 is then converted to the bromide via anucleophilic displacement reaction with sodium bromide as described byWilt, J., J. Org. Chem., 35 (8), 1970, pp. 2803-2806 to yield compound63.

Alternatively, a one step procedure is provided in Hooz, J. et al., Can.J. Chem., 46, 1968, pp. 86-87.

Compound 63 can then be reacted with diethylene triamine (available fromthe Aldrich Chemical company) by the following procedure.

Compound 63 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and thediethylene triamine (0.283 moles), which is available from the AldrichChemical Company is added while stirring is continued. The resultantsolution is heated to reflux overnight under nitrogen. This solution isthen allowed to cool to room temperature and is poured onto distilled H₂O. Then, this mixture is concentrated in vacuo until only the H₂ Oremains. An additional 50-75 ml H₂ O is added and the reaction mixtureis cooled to 0° C. The solid is filtered and washed with ice cold water.This material is then redissolved in EtOH and precipitated with 10 NHCl. After filtration of this suspension, the resultant solid isrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop is also collected from the second filtrate upon standing andby precipitation with EtOH from the first filtrate. These solids arethen combined and the final product 64 is obtained and is subjected tohigh vacuum overnight. Characterization can be effected by calculatingthe molecular mass of the free base amine from the exact isotopic massformulas well known to those skilled in the art and comparing theresultant mass with that obtained by a high resolution mass spectrometrymolecular weight determination such as are well known to those skilledin the art.

Amine derivatives of compound 63 can be synthesized as follows. Compound63 (0.0081 moles) is added to a 250 ml round bottom flask equipped witha magnetic stir bar and reflux condenser. Methanol (150 ml) is thenadded to this flask while stirring under nitrogen and the appropriateamine selected from the following list (0.283 moles), which areavailable from the Aldrich Chemical Company, is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product 30a-30o is obtainedand is subjected to high vacuum overnight.

Listed below are the compounds that can be obtained from theircorresponding amines.

4-amino-1-benzylpiperidine to yield 63a

spermine to yield to yield 63b

pyridine to yield to yield 63c

2-(2-Aminoethyl)-1-methylpyrrolidine to yield 63d

1-(2-Aminoethyl)pyrrolidine to yield 63e

1-(2-Aminoethyl)piperidine to yield 63f

2-(2-Aminoethyl)pyridine to yield 63g

1-(2-Aminoethyl)piperazine to yield 63h

4-(2-Aminoethyl)morpholine to yield 63i

1-Amino4-(2-hydroxyethyl)piperazine to yield 63j

4-(Aminomethyl)piperidine to yield 63k

2-(Aminomethyl)pyridine to yield 63l

aniline to yield 63m

1-(3-Aminopropyl)imidazole to yield 63n

4-(3-Aminopropyl)morpholine to yield 63o

Characterization can be effected by calculating the molecular mass ofthe free base amine from the exact isotopic mass formulas well known tothose skilled in the art and comparing the resultant mass with thatobtained by a high resolution mass spectrometry molecular weightdetermination such as are well known to those skilled in the art. Notethat no hydrolysis is necessary.

Since the structures of compounds 63a-63o are unambiguous from the abovegeneric procedures, their structures are not shown.

EXAMPLE 18

Synthesis of Compounds 68 and 68a-68p

The reaction scheme for the general synthesis of compounds 68, 68a, andtheir precursors, is given in FIG. 24. The structure formula ofcompounds 68a-68p are given in FIG. 25.

2-Methylbenzoxazole 59 is obtained from the Aldrich Chemical Company(Milwaukee, Wis.). It is alkylated to produce compound 65 using3-brom-1-propanol by adapting procedures of Gaugain et al.,Biochemistry, 17 (24), 1978, pp. 5071-5078. Compound 45 is obtained fromthe Aldrich Chemical Company and is reacted with 3-bromo-1-propanol(also available from the Aldrich Chemical Company) using procedures suchas found in Gaugain et al., Biochemistry, 17 (24) 1978, 5071-5078. Thecondensation of compound 65 and 46 is effected by adapting proceduresfound in Hamer, Francis, "Heterocyclic Compounds, Cyanine Dyes andRelated Compounds", Wiley, 1964, p. 37 to yield compound 60. Compound 67is then obtained by converting the alcohol to the tosylate by usingprocedures such as found in Wiberg, K. et al., J. Am. Chem. Soc., 92(3), 1970, pp. 553-564. The tosylate 67 is then converted to the bromidevia a nucleophilic displacement reaction with sodium bromide as perWilt, J., J. Org. Chem., 35 (8), 1970, pp. 2803-2806 to yield compound68.

Alternatively, a one step procedure, as provided by Hooz, J. et al.,Can. J. Chem., 46, 1968, pp. 86-87, can be used.

Compound 68 can then be reacted with diethylene triamine or otherappropriate amine (available from the Aldrich Chemical Company) by thefollowing procedure.

Compound 68 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and anamine, such as diethylene triamine or other appropriate base (0.283moles), which is available from the Aldrich Chemical Company is addedwhile stirring is continued. The resultant solution is heated to refluxovernight under nitrogen. This solution is then allowed to cool to roomtemperature and is poured onto distilled H₂ O. Then, this mixture isconcentrated in vacuo until only the H₂ O remains. An additional 50-75ml H₂ O is added and the reaction mixture is cooled to 0° C. The solidis filtered and washed with ice cold water. This material is thenredissolved in EtOH and precipitated with 10 N HCl. After filtration ofthis suspension, the resultant solid is recrystallized from hot ethanolupon cooling to 0° C. for 15 minutes. A second crop is also collectedfrom the second filtrate upon standing and by precipitation with EtOHfrom the first filtrate. These solids are then combined and the finalproduct 68a is obtained and is subjected to high vacuum overnight.Characterization is effected by calculating the molecular mass of thefree base amine from the exact isotopic mass formulas well known tothose skilled in the art and comparing the resultant mass with thatobtained by a high resolution mass spectrometry molecular weightdetermination such as are well known to those skilled in the art.

As long as the ratios of reagents and solvents are held constant, onecan scale up or down the amounts of reagents using the method of ratioand proportions well known to those skilled in the art.

Listed below are products that can be obtained from their correspondingamines.

diethylene triamine to yield compound 68a

4-amino-1-benzylpiperidine to yield compound 68b

spermine to yield compound 68c

pyridine to yield compound 68d

2-(2-Aminoethyl)-1-methylpyrrolidine to yield compound 68e

1-(2-Aminoethyl)pyrrolidine to yield compound 68f

1-(2-Aminoethyl)piperidine to yield compound 68g

2-(2-Aminoethyl)pyridine to yield compound 68h

1-(2-Aminoethyl)piperazine to yield compound 68i

4-(2-Aminoethyl)morpholine to yield compound 68j

1-Amino4-(2-hydroxyethyl)piperazine to yield 68k

4-(Aminomethyl)piperidine to yield 68l

2-(Aminomethyl)pyridine to yield 68m

aniline to yield 68n

1-(3-Aminopropyl)imidazole to yield 68o

4-(3-Aminopropyl)morpholine to yield 68p

EXAMPLE 19

Synthesis of Compounds 71 and 71a-71p

The reaction scheme for the general synthesis of compounds 71, 71a, andtheir precursors, is given in FIG. 26. The structure formula ofcompounds 71a-71p are given in FIG. 27.

2-Methylbenzoxazole 59 is obtained from the Aldrich Chemical Company(Milwaukee, Wis.). It is alkylated to produce compound 65 using3-bromo-1-propanol by adapting procedures such as found in Gaugain, et.al, Biochemistry, Vol. 17, No. 24, 1978, pp. 5071-5078 to obtaincompound 13. Compound 45 is obtained from the Aldrich Chemical Companyand is reacted with methyl iodide (also available from the AldrichChemical Company) using procedures such as found in Southwick et al.U.S. Pat. No. 4,981,977, Jan. 1, 1991 or in Ernst et al., Cytometry, 10,1989, pp. 3-10 to yield compound 55. The condensation of compound 65 and55 is effected by adapting procedures found in Hamer, Francis,"Heterocyclic Compounds, Cyanine Dyes and Related Compounds", Wiley,1964, p. 37 to yield compound 69. Compound 70 is then obtained byconverting the alcohol to the tosylate by using procedures such as foundin Wiberg, K. et al., J. Am. Chem. Soc., 92 (3), 1970, pp. 553-564. Thetosylate 70 can then be converted to the bromide via a nucleophilicdisplacement reaction with sodium bromide as disclosed by Wilt, J., J.Org. Chem., 35 (8), 1970, pp. 2803-2806 to yield compound 20.

Alternatively, a one step procedure, as provided by Hooz, J. et al.,Can. J. Chem., 46, 1968, pp. 86-87, can be used. Compound 70 can then bereacted with diethylene triamine or other appropriate amine (availablefrom the Aldrich Chemical company) by the following procedure.

Compound 71 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen anddiethylene triamine or other appropriate amine, (0.283 moles), which isavailable from the Aldrich Chemical Company is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the final product 71a-71p is obtainedand is subjected to high vacuum overnight. Characterization can beeffected by calculating the molecular mass of the free base amine fromthe exact isotopic mass formulas well known to those skilled in the artand comparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

As long as the ratios of reagents and solvents are held constant, onecan scale up or down the amounts of reagents using the method of ratioand proportions well known to those skilled in the art.

Listed below are products that can be obtained for their correspondingamines.

diethylene triamine to yield compound 20a

4-amino-i-benzylpiperidine to yield compound 20b

spermine to yield compound 20c

pyridine to yield compound 20d

2-(2-Aminoethyl)-1-methylpyrrolidine to yield compound 20e

1-(2-Aminoethyl)pyrrolidine to yield compound 20f

1-(2-Aminoethyl)piperidine to yield compound 20g

2-(2-Aminoethyl)pyridine to yield compound 20h

1-(2-Aminoethyl)piperazine to yield compound 20i

4-(2-Aminoethyl)morpholine to yield compound 20j

1-Amino4-(2-hydroxyethyl)piperazine to yield 20k

4-(Aminomethyl)piperidine to yield 20l

2-(Aminomethyl)pyridine to yield 20m

aniline to yield 20n

1-(3-Aminopropyl)imidazole to yield 20o

4-(3-Aminopropyl)morpholine to yield 20p

EXAMPLE 20

Synthesis of Compound 80 and its Related Compounds

The reaction scheme for the general synthesis of compound 80 and itsprecursors is given in FIG. 28.

Compound 72 is obtained from the Aldrich Chemical Company. Compound 73can be synthesized from compound 72, compound 74 can be synthesized fromcompound 73, and compound 75 can be synthesized from compound 74, eachfollowing the procedure of Dervan et al., J. Am. Chem. Soc., Vol. 100,No. 6, 1978, pp. 1968-1970 or secondary references contained within.

Compound 76 is synthesized from compound 75 by the procedure of Gaugain,et. al, Biochemistry, Vol. 17, No. 24, 1978, pp. 5071-5078. Compound 77can be synthesized from compound 76 by the procedure of Dervan, P. B.,Becker, M. M., J. Am. Chem. Soc., 1978, Vol. 100, No. 6, 1968-1970 orsecondary references contained within. Compound 78 can be synthesizedfrom compound 77 by the procedures such as Lee et al., J. Am. Chem.Soc., 88 (14), 1966, pp. 3440-3441 or references contained within.Compound 79 can be synthesized by the following procedure. Compound 78(0.0081 moles) is added to a 250 ml round bottom flask equipped with amagnetic stir bar and reflux condenser. Methanol (150 ml) is then addedto this flask while stirring under nitrogen and the diethylene triamine(0.283 moles), which is available from the Aldrich Chemical Company isadded while stirring is continued. The resultant solution is heated toreflux overnight under nitrogen. This solution is then allowed to coolto room temperature and is poured onto distilled H₂ O. Then, thismixture is concentrated in vacuo until only the H₂ O remains. Anadditional 50-75 ml H₂ O is added and the reaction mixture is cooled to0° C. The solid is filtered and washed with ice cold water. Thismaterial is then redissolved in EtOH and precipitated with 10 N HCl.After filtration of this suspension, the resultant solid isrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop is also collected from the second filtrate upon standing andby precipitation with EtOH from the first filtrate. These solids arethen combined and the product 79 is obtained and is subjected to highvacuum overnight. Characterization is effected by calculating themolecular mass of the free base amine from the exact isotopic massformulas well known to those skilled in the art and comparing theresultant mass with that obtained by a high resolution mass spectrometrymolecular weight determination such as are well known to those skilledin the art. The final compound 80 can be obtained by using the reductionprocedure of Dervan, P. B., Becker, M. M., J. Am. Chem. Soc., 1978, 100(6), 1968-1970. Characterization and purification of all intermediatescan be accomplished using methods well known to those skilled in theart.

Amine derivatives of compound 78 can be synthesized as follows. Compound78 (0.0081 moles) is added to a 250 ml round bottom flask equipped witha magnetic stir bar and reflux condensor. Methanol (150 ml) is thenadded to this flask while stirring under nitrogen and the appropriateamine selected from the following list (0.283 moles), which areavailable from the Aldrich Chemical Company, is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the product 78a-78o is obtained andis subjected to high vacuum overnight. Characterization can be effectedby calculating the molecular mass of the free base amine from the exactisotopic mass formulas well known to those skilled in the art andcomparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

Given below are the compounds that can be obtained from theircorresponding amines.

4-amino-1-benzylpiperidine to yield 78a

spermine to yield to yield 78b

pyridine to yield to yield 78c

2-(2-Aminoethyl)-1-methylpyrrolidine to yield 78d

1-(2-Aminoethyl)pyrrolidine to yield 78e

1-(2-Aminoethyl)piperidine to yield 78f

2-(2-Aminoethyl)pyridine to yield 2k

1-(2-Aminoethyl)piperazine to yield 78h

4-(2-Aminoethyl)morpholine to yield 78i

1-Amino4-(2-hydroxyethyl)piperazine to yield 78j

4-(Aminomethyl)piperidine to yield 78k

2-(Aminomethyl)pyridine to yield 78l

aniline to yield 78m

1-(3-Aminopropyl)imidazole to yield 78n

4-(3-Aminopropyl)morpholine to yield 78o

Synthesis and purification of the final product 78aa-78oo can beaccomplished by the following protocol to yield the corresponding finalproducts as shown below. The nitro compound 78a-78o can be converted tothe appropriate amine using the procedure of Dervan et al., J. Am. Chem.Soc., 100 (6), 1978, pp. 1968-1970.

Listed below are the products that can be obtained from theircorresponding immediate precursors.

78a to yield 78aa

78b to yield to yield 78bb

78c to yield to yield 78cc

78d to yield 78dd

78e to yield 78ee

78f to yield 78ff

78g to yield 78gg

78h to yield 78hh

78i to yield 78ii

78j to yield 78jj

78k to yield 78kk

78l to yield 78ll

78m to yield 78mm

78n to yield 78nn

78o to yield 78oo

Characterization can be effected by calculating the molecular mass ofthe free base amine from the exact isotopic mass formulas well known tothose skilled in the art and comparing the resultant mass with thatobtained by a high resolution mass spectrometry molecular weightdetermination such as are well known to those skilled in the art.

Since the structures of compounds 78a-78o and 78aa-78oo are unambiguousfrom the given generic procedures, their structures are not shown.

EXAMPLE 21

Synthesis of Compound 85 and its Related Compounds

The reaction scheme for the general synthesis of compound 85 and itsprecursors is given in FIG. 29.

Starting material 81 (0.0876 moles) is obtained from the AldrichChemical Company (Milwaukee, Wis.) and is added to a single neck 3.0liter round bottom flask under Argon and equipped with a magnetic stirbar and a reflux condenser. To this vessel, 1.0 liter of dry pyridine isadded while stirring. Stirring of the resulting suspension is continuedfor 15 minutes until all the solid is dissolved. A catalytic amount ofN,N-dimethylaminopyridine (1.07 g, 0.00876 moles) is added to thissolution while stirring. Acetic anhydride (462 g, 4.9 moles) is thenadded and the resulting reaction mixture is refluxed for 8-12 hours. Thereaction mixture is then allowed to cool and the solvent is removed invacuo. For purification of the product 42, a gradient silica gel columnis performed using an appropriate solvent system determined usingmethods well known to those skilled in the art such as Thin LayerChromatography. Fractions of 10.0 ml are collected and appropriatefractions are recombined and the solvent is removed in vacuo. Theresidue is then dissolved in hot EtOH (220 ml) and precipitated bycooling to 0° C. The mother liquor is decanted off and 200 ml of freshEtOH is added. The solid is redissolved by heating and is allowed tocrystallize and -4° C. for 48 hours. Crystals are collected from boththe mother liquor and the second recrystalization and are washed with asmall amount of cold EtOH and dried under high vacuum for several hours.The resultant compound 82 can be characterized by high resolution massspectrometry as well known to those skilled in the art.

Compound 83 can be synthesized from the imide 42 via a modification of aliterature procedure of Gaugain, et al., Biochemistry, Vol. 17, No. 24,1978, pp. 5071-5078 for quarternization of the diamide of 3,8diamino-6-phenyl phenanthridine. Imide 82 (0.023 moles) is placed in a2.0 liter round bottom flask under Argon and is equipped with a magneticstir bar and reflux condenser. 1,3-Dibromopropane (1.0 liter, 9.86moles) is added to this flask and the resultant mixture is brought toreflux for about 7 hours. The solution is cooled overnight and theprecipitant is filtered and washed with Et₂ O. This material isrecrystallized from CH₃ OH to yield diacetyl bromide 83 which can becharacterized by mass spectrometry and other methods known to thoseskilled in the art.

Compound 83 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and thediethylene triamine (0.283 moles), which is available from the AldrichChemical Company is added while stirring is continued. The resultantsolution is heated to reflux overnight under nitrogen. This solution isthen allowed to cool to room temperature and is poured onto distilled H₂O. Then, this mixture is concentrated in vacuo until only the H₂ Oremains. An additional 50-75 ml H₂ O is added and the reaction mixtureis cooled to 0° C. The solid is filtered and washed with ice cold water.This material is then redissolved in EtOH and precipitated with 10 NHCl. After filtration of this suspension, the resultant solid isrecrystallized from hot ethanol upon cooling to 0° C. for about 15minutes. A second crop is also collected from the second filtrate uponstanding and by precipitation with EtOH from the first filtrate. Thesesolids are then combined and the product 84 can be obtained and can besubjected to high vacuum overnight. Characterization can be effected bycalculating the molecular mass of the free base amine from the exactisotopic mass formulas well known to those skilled in the art andcomparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

Synthesis and purification of the final product 85 can be accomplishedby the following protocol. The imide (0.0036 moles) 84 is dissolved in75.0 ml methanol and 75 ml of 4N HCl is added. The mixture is refluxedfor about 2 hours and allowed to cool. Ethanol is added to this solutionand resulting precipitate is filtered and washed with a minimal amountof cold ethanol. The filtrate is reconcentrated and fresh ethanol andconcentrated aqueous HCl is added. This resulting precipitate is alsofiltered. Next, this filtrate is concentrated to near dryness and Et₂ Ois added and the solid filtered off. The last remaining unfilterableresidue is then dissolved in concentrated HCl and precipitated withEtOH. This material is filtered and washed with Ethanol. All solidmaterials are combined from the above sequence and is subjected to highvacuum overnight to obtain 85. Characterization can be effected bycalculating the molecular mass of the free base amine from the exactisotopic mass formulas well known to those skilled in the art andcomparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

Amine derivatives of compound 83 can be synthesized as follows: Compound83 (0.0081 moles) is added to a 250 ml round bottom flask equipped witha magnetic stir bar and reflux condenser. Methanol (150 ml) is thenadded to this flask while stirring under nitrogen and the appropriateamine selected from the following list (0.283 moles), which areavailable from the Aldrich Chemical Company, is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the second filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the product 83a-83o can be obtainedand is subjected to high vacuum overnight. Characterization can beeffected by calculating the molecular mass of the free base amine fromthe exact isotopic mass formulas well known to those skilled in the artand comparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

Listed below are the products that can be obtained from theircorresponding amines.

4-amino-1-benzylpiperidine to yield 83a

spermine to yield to yield 83b

pyridine to yield to yield 83c

2-(2-Aminoethyl)-1-methylpyrrolidine to yield 83d

1-(2-Aminoethyl)pyrrolidine to yield 83e

1-(2-Aminoethyl)piperidine to yield 83f

2-(2Aminoethyl)pyridine to yield 83g

1-(2-Aminoethyl)piperazine to yield 83h

4-(2-Aminoethyl)morpholine to yield 83i

1-Amino4-(2-hydroxyethyl)piperazine to yield 83j

4-(Aminomethyl)piperidine to yield 83k

2-(Aminomethyl)pyridine to yield 83l

aniline to yield 83m

1-(3-Aminopropyl)imidazole to yield 83n

4-(3-Aminopropyl)morpholine to yield 83o

Synthesis and purification of the final product 83aa-83oo can beaccomplished by the following protocol to yield the corresponding finalproducts as shown below. The imide (0.0036 moles) 83a-83o is dissolvedin 75.0 ml methanol and 75 ml of 4N HCl is added. The mixture isrefluxed for about 2 hours and allowed to cool. Ethanol is added to thissolution and resulting precipitate is filtered and washed with a minimalamount of cold ethanol. The filtrate is reconcentrated and fresh ethanoland concentrated aqueous HCl is added. This resulting precipitate isalso filtered. Next, this filtrate is concentrated to near dryness andEt₂ O is added and the solid filtered off. The last remainingunfilterable residue is then dissolved in concentrated HCl andprecipitated with EtOH. This material is filtered and washed withEthanol. All solid materials are combined from the above sequence and issubjected to high vacuum overnight to obtain 83aa-83oo. Characterizationcan be effected by calculating the molecular mass of the free base aminefrom the exact isotopic mass formulas well known to those skilled in theart and comparing the resultant mass with that obtained by a highresolution mass spectrometry molecular weight determination such as arewell known to those skilled in the art.

Listed below are the products that can be obtained from theircorresponding immediate starting materials.

83a to yield 83aa

83b to yield 83bb

83c to yield 83cc

83d to yield 83dd

83e to yield 83ee

83f to yield 83ff

83g to yield 83gg

83h to yield 83hh

83i to yield 83ii

83j to yield 83jj

83k to yield 83kk

83l to yield 83ll

83m to yield 83mm

83n to yield 83nn

83o to yield 83oo

Since the structures of compounds 83a-83o and 83aa-83oo are unambiguousfrom the generic procedures, their structures are not given.

EXAMPLE 22

Synthesis of Compound 90 and its Related Compounds

The reaction scheme for the general synthesis of compound 90 and itsprecursors is given in FIG. 30.

Starting material 86 (0.0876 moles) is obtained from the AldrichChemical Company (Milwaukee, Wis.) and is added to a single neck 3.0liter round bottom flask under Argon and equipped with a magnetic stirbar and a reflux condenser. Acetic anhydride (462 g, 4.9 moles) is thenadded and the resulting reaction mixture is refluxed for 8-12 hours. Thereaction mixture is then allowed to cool and the solvent is removed invacuo. For purification of the product 87, a gradient silica gel columnis performed using an appropriate solvent system determined usingmethods well known to those skilled in the art. Fractions of 10.0 ml arecollected and appropriate fractions are recombined and the solvent isremoved in vacuo. The residue is then dissolved in hot EtOH (220 ml) andprecipitated by cooling to 0° C. The mother liquor is decanted off and200 ml of fresh EtOH is added. The solid is redissolved by heating andis allowed to crystallize and -4° C. for 48 hours. Crystals arecollected from both the mother liquor and the second recrystalizationand are washed with a small amount of cold EtOH and dried under highvacuum for several hours. The resultant compound 87 can be characterizedby high resolution mass spectrometry as well known to those skilled inthe art.

Compound 88 can be synthesized from the diamide 87 via a modification ofa literature procedure of Gaugain, et al., Biochemistry, Vol. 17, No.24, 1978, pp. 5071-5078 for quarternization of the diamide of3,8-diamino-6-phenyl phenanthridine. Diamide 87 (0.023 moles) is placedin a 2.0 liter round bottom flask under Argon and is equipped with amagnetic stir bar and reflux condenser. 1,3-Dibromopropane (1.0 liter,9.86 moles) is added to this flask and the resultant mixture is broughtto reflux for about 7 hours. The solution is cooled overnight and theprecipitant is filtered and washed with Et₂ O. This material can berecrystallized from CH₃ OH to yield diacetyl bromide 88 which can becharacterized by mass spectrometry and other methods known to thoseskilled in the art.

Compound 88 (0.0081 moles) is added to a 250 ml round bottom flaskequipped with a magnetic stir bar and reflux condenser. Methanol (150ml) is then added to this flask while stirring under nitrogen and thediethylene triamine (0.283 moles), which is available from the AldrichChemical Company, is added while stirring is continued. The resultantsolution is heated to reflux overnight under nitrogen. This solution isthen allowed to cool to room temperature and is poured onto distilled H₂O. Then, this mixture is concentrated in vacuo until only the H₂ Oremains. An additional 50-75 ml H₂ O is added and the reaction mixtureis cooled to 0° C. The solid is filtered and washed with ice cold water.This material is then redissolved in EtOH and precipitated with 10 NHCl. After filtration of this suspension, the resultant solid isrecrystallized from hot ethanol upon cooling to 0° C. for 15 minutes. Asecond crop is also collected from the second filtrate upon standing andby precipitation with EtOH from the first filtrate. These solids arethen combined and the product 89 can be obtained and is subjected tohigh vacuum overnight. Characterization can be effected by calculatingthe molecular mass of the free base amine from the exact isotopic massformulas well known to those skilled in the art and comparing theresultant mass with that obtained by a high resolution mass spectrometrymolecular weight determination such as are well known to those skilledin the art.

Synthesis and purification of the final product 90 can be accomplishedby the following protocol. The diamide (0.0036 moles) 89 is dissolved in75.0 ml methanol and 75 ml of 4N HCl is added. The mixture is refluxedfor 2 hours and allowed to cool. Ethanol is added to this solution andresulting precipitate is filtered and washed with a minimal amount ofcold ethanol. The filtrate is reconcentrated and fresh ethanol andconcentrated aqueous HCl is added. This resulting precipitate is alsofiltered. Next, this filtrate is concentrated to near dryness and Et₂ Ois added and the solid filtered off. The last remaining unfilterableresidue is then dissolved in concentrated HCl and precipitated withEtOH. This material is filtered and washed with Ethanol. All solidmaterials are combined from the above sequence and subjected to highvacuum overnight to obtain compound 90. Characterization can be effectedby calculating the molecular mass of the free base amine from the exactisotopic mass formulas well known to those skilled in the art andcomparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

Amine derivatives of compound 88 can be synthesized as follows: Compound88 (0.0081 moles) is added to a 250 ml round bottom flask equipped witha magnetic stir bar and reflux condenser. Methanol (150 ml) is thenadded to this flask while stirring under nitrogen and the appropriateamine selected from the following list (0.283 moles), which areavailable from the Chemical Company, is added while stirring iscontinued. The resultant solution is heated to reflux overnight undernitrogen. This solution is then allowed to cool to room temperature andis poured onto distilled H₂ O. Then, this mixture is concentrated invacuo until only the H₂ O remains. An additional 50-75 ml H₂ O is addedand the reaction mixture is cooled to 0° C. The solid is filtered andwashed with ice cold water. This material is then redissolved in EtOHand precipitated with 10 N HCl. After filtration of this suspension, theresultant solid is recrystallized from hot ethanol upon cooling to 0° C.for 15 minutes. A second crop is also collected from the 2^(nd) filtrateupon standing and by precipitation with EtOH from the first filtrate.These solids are then combined and the product 48a-48o is obtained andis subjected to high vacuum overnight. Characterization can be effectedby calculating the molecular mass of the free base amine from the exactisotopic mass formulas well known to those skilled in the art andcomparing the resultant mass with that obtained by a high resolutionmass spectrometry molecular weight determination such as are well knownto those skilled in the art.

By combining the generic procedure for the synthesis of phenanthridiniumderivatives 28a-42a and 28b-42b with the above procedures for thesynthesis of bromide compound 88, 83, 78, 63 or 49, any combination ofamine tail--intercalator molecular segments using the generic procedurefor alkylation of bromides with amines as already described above can besynthesized, except that for derivatives of compounds 49 and 63, nohydrolysis step is need so the hydrolysis step is eliminated. In thecase of derivatives of compound 78 no hydrolysis step will be needed buta reduction step is needed according to the procedure of Dervan et al.,J. Am. Chem. Soc., 100 (6), 1978, pp. 1968-1970.

EXAMPLE 23

General Procedure to Form a "Matrix" of Compounds

Synthesis and purification of the final product 88aa-88oo can beaccomplished by the following protocol to yield the corresponding finalproducts as shown below. The imide (0.0036 moles) 88a-88o is dissolvedin 75.0 ml methanol and 75 ml of 4N HCl is added. The mixture isrefluxed for 2 hours and allowed to cool. Ethanol is added to thissolution and resulting precipitate is filtered and washed with a minimalamount of cold ethanol. The filtrate is reconcentrated and fresh ethanoland concentrated aqueous HCl is added. This resulting precipitate isalso filtered. Next, this filtrate is concentrated to near dryness andEt₂ O is added and the solid filtered off. The last remainingunfilterable residue is then dissolved in concentrated HCl andprecipitated with EtOH. This material is filtered and washed withEthanol. All solid materials are combined from the above sequence and issubjected to high vacuum overnight to obtain 88aa-88oo. Characterizationcan be effected by calculating the molecular mass of the free base aminefrom the exact isotopic mass formulas well known to those skilled in theart and comparing the resultant mass with that obtained by a highresolution mass spectrometry molecular weight determination such as arewell known to those skilled in the art.

Listed below are the products that can be obtained from theircorresponding immediate precursors.

88a to yield 88aa

88b to yield 88bb

88c to yield 88cc

88d to yield 88dd

88e to yield 88ee

88f to yield 88ff

88g to yield 88gg

88h to yield 88hh

88i to yield 88ii

88j to yield 88jj

88k to yield 88kk

88l to yield 88ll

88m to yield 88mm

88n to yield 88nn

88o to yield 88oo

Since the structures of compounds 88a-88o and 88aa-88oo are unambiguousfrom the generic procedures, their structures are not shown.

EXAMPLE 24

Hybridization Assay Using Compounds 24-27

In four separate experiments, PTA 24, compound 25, compound 26, andcompound 27 were used to quantitate hybridization when a targetoligonucleotide was titrated with its complementary partner. By alsoconducting three simultaneous but independent experiments, a comparisonof ethidium bromide staining, propidium iodide staining and ethidiumhomodimer staining verus PTA 24 was made as follows. Results can befound in FIG. 31. Results of a comparison of ethidium bromide staining,compound 24 (PTA) staining, compound 25 staining, compound 26 stainingand compound 27 staining, as per the following protocol, are found inFIG. 32. Complementary strands of DNA oligodeoxythymidylic acid, d(pT)₉,and oligodeoxyadenylic acid, (d(pA)₉), were obtained from the SigmaChemical Co. in St. Louis, Mo. A stock solution of d(pA)₉ was made at5.0 units/0.5 ml of 0.004 M TRIS, 0.001 M EDTA, pH 8.2 buffer. ForpolyA, ε=8.4 AU/mM cm or 8400 M⁻¹ cm⁻¹ ; Therefore, with 9 base pairsfor d(pA)₉, the ε is 75,600 M⁻¹ cm⁻¹. This stock was then diluted 100×to obtain stock at 6.61×10⁻⁷ M, or 0.66 μM. The d(pT)9 stock was made atunits/5.0 ml and used for titration after dilution 10× with the samebuffer. Since the ε for polyT is 8.15 AU/mM cm or 8,150 M⁻¹ cm⁻¹ perbase pair, or 73,350 M⁻¹ cm⁻¹ per oliogo, the concentration of the oligostock was 6.8 μM in DNA molecules. A titration was performed using aHitachi F-4010 Fluorescence Spectrophotometer using polystyrenedisposable 4.0 ml cuvettes to obtain a fully corrected spectra and anexcitation wavelength of 488-550 nm (using 488 nm for this experiment)and an emission wavelength of 600-650 nm (using 625 nm for thisexperiment). Equivalents of d(pT)₉ were added at the followingincrements: 0.020, 0.080, 0.150, 0.300, 0.500, 0.700, 1.000, 2.000,5.000 equivalents. Each sample in the titration curve was preparedindividually by dividing the initial d(TA)₉ stock in 10× 1.0 mlincrements. The addition of complement was then accomplished bymicropipetting an appropriate amount (2, 5, 8, 15, 30, 50, 70, 100, 200,and 500 μl, respectively) of d(pT)9 stock to each of a series of the 10aliquots. Each aliquout, containing progressively larger molar ratios ofthe two complementary strands, was incubated a ambient temperature for 1hr 45 minutes, the corresponding dye was added as 100.0 μl aliquots of a15 μM solution of the corresponding dye in 0.004M TRIS, 0.001M EDTA, pH8.2 buffer. This corresponds to a dye/DNA b.p. ratio of 1/4 atsaturation with complementary oligo. Overall concentrations of dye andoligo vary in the saturation plot because of the use of variedincrements additions from the same stock solution. After an additional45 minutes incubation time after addition of the correspondingappropriate dye, the relative fluorescence intensity was read at 625 nmand recorded to generate a standard curve which is directly proportionalto the quantity of dsDNA hybridization, or target sequence, under thesame conditions. The background, or initial residual fluorescence, isthen substrated out as a constant for all curves for comparison of thevarious titration curves on the same graphs.

It is apparent to those skilled in the art that the high bindingaffinity of the intercalator compounds 24, 25, 26, or 27 of the presentinvention is especially useful relative to conventional intercalatorssuch as ethidium bromide (FIG. 32). In FIG. 32 (EXAMPLE 24), thesensitivity or response to ds-DNA concentration in the presence ofintercalator compounds 24, 25, 26, and 27 is shown compared to ethidiumbromide. When concentrations of oligonucleotide are less than 10⁻⁶ M,the advantages of such high affinity intercalators is especiallyapparent.

In comparison of PTA 24 to homobifunctional intercalators such asethidium homodimer (FIG. 32), again a clear advantage is sensitivity isobserved. In this case, the increased sensitivity may be related toreduced self-quenching in the PTA 24 as well as its high affinity forDNA.

In FIG. 8 and FIG. 9, the concentration of ds-DNA was higher and thedifferential between the ethidium bromide and PTA 24 (FIG. 9) andethidium homodimer and PTA 24 (FIG. 8) was not significant because ofthe higher concentration of DNA used.

In summary, the present invention offers clear advantages in allowingthe detection of ds-DNA hybridization at much lower concentrations thanconventional staining methods currently available or known in the art.

EXAMPLE 25

Hybridization Assay/Using Intercalator Compound 1, 2, 3, 24, 25, 26, 27,28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b, 36b, 37b, 38, 39b, 40b, 41b,42b, or 80

DNA Intercalator 1, 2, 1, 24, 25, 26, 27, 28b, 29b, 30b, 31b, 32b, 33b,34b, 35b, 36b, 37b, 38, 39b, 40b, 41b, 42b, or 80 can be used toquantitate hybridization when a target oligonucleotide is titrated withits complementary partner. Complementary strands of DNAoligodeoxythymidylic acid, d(pT)₉, and oligodeoxyadenylic acid,(d(pA)₉), can be obtained from the Sigma Chemical Co. in St. Louis, Mo.A stock solution of d(pA)9 is made at 5 units/ml of 0.05M TRIS, 0.2NNaCl, 1 mM EDTA, pH 8.4 or other suitable buffer. For polyA, ε=8.4 AU/mMcm or 8,400 M⁻¹ cm⁻¹ ; therefore, with 9 base pairs for d(pA)9, the ε is75,600 M⁻¹ cm⁻¹. This stock is then diluted to obtain stock from0.066-66 μM. The d(pT)₉ stock is made at 25 units/5.0 ml and used fortitration without further dilution in the same buffer. Since the ε forpolyT is 8.15 AU/mM cm or 8,150 M-1cm⁻¹ per base pair, or 73,350 M-1cm⁻¹per oligo, the concentration of the oligo stock is 0.0068-660 μM in DNAmolecules. A titration can be performed using a FluorescenceSpectrophotometer using an excitation wavelength of 488-550 nm (optimalaround 534) and an emission wavelength of 600-650 nm (optimal around625). Equivalents of d(pT)₉ is added at the following increments: 0.02,0.05, 0.080, 0.150, 0.300, 0.500, 0.700, 1.00, 2.00, 5.00 equivalents.Each sample in the titration curve is prepared individually by dividingthe initial d(pA)9 stock into 10×1.0 ml aliquots. The addition ofcomplement is then accomplished by micropipetting an appropriate amount(2, 5, 8, 15, 30, 50, 70, 100, 200, and 500 μl, respectively) of d(pT)9stock to each of a series of the 10 aliquots when the d(pT)9 stock is10× the concentration of the d(pA)9 stock. Each aliquot, obtainingprogressively larger molar ratios of the two complementary strands, isincubated at ambient temperature for 0.25-2.5 hours, the dye is added as1-500 μl aliquots of a 1-1000 μM solution of the dye in 0.05M TRIS, 0.2NNaCl, 1 mM EDTA, pH 8.4 buffer or other suitable buffer. Thiscorresponds to a dye/DNA b.p. ratio of 1/1-1/1000 at saturation withcomplementary oligo. Overall concentrations of dye and oligo vary in thesaturation plot because of the use of varied increments additions fromthe same stock solution. After an additional 15 minute incubation time,the relative fluorescence intensity is then read at between 580-680 nmand recorded to generate a standard curve which is directly proportionalto the quantity of dsDNA hybridization, or target sequence, under theseconditions.

EXAMPLE 26

Determination of Hybridization of Complementary Binding Pairs Other Thand(pT)9 and d(dA)9 Using Intercalator Compound 1, 2, 3, 24, 25, 26, 27,28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b, 36b, 37b, 38, 39b, 40b, 41b,42b, or 80

By using the above described procedure from EXAMPLE 25, intercalatorcompound 1, 2, 3, 24, 25, 26, 27, 28b, 29b, 30b, 31b, 32b, 33b, 34b,35b, 36b, 37b, 38, 39b, 40b, 41b, 42b, or 80 can be used to determinethe quantity of hybridization of any other complementary DNA strands bysubstituting d(pT)9 with the appropriate complementary DNA andsubstituting d(pA)9 with the appropriate target DNA at appropriateconcentrations that are determined by one skilled in the art anddepending on the degree of complementary regions that are expected as isdetermined by one skilled in the art. In each case, the excitationwavelength of 450-550 nm (optimal at 534 nm) can be used and therelative fluorescence intensity is then read at between 580-680 nm andrecorded to generate a standard curve which is directly proportional tothe quantity of dsDNA hybridization, or target sequence, under theseconditions.

EXAMPLE 27

Hybridization Assay Using Intercalator Compound 7, 8, 50, 54a, 54b, 54c,54d, 54e, 54f, 54g, 54h, 54i, 54j, 54k, 54l, 54m, 54n, 54o, 54p, 58a,58b, 58c, 58d, 58e, 58f, 58g, 58h, 58i, 58j, 58k, 58l, 58m, 58n, 58o,58p, 64, 68a, 68b, 68c, 68d, 68e, 68f, 68g, 68h, 68i, 68j, 68k, 68l,68m, 68n, 68o, 68p, 71a, 71b, 71c, 71d, 71e, 71f, 71g, 71h, 71i, 71j,71k, 71l, 71m, 71n, 71o, or 71p

By using the above procedure in EXAMPLE 26, the hybridization of any twocomplementary strands can be determined using intercalator compound 7,8, 50, 54a, 54b, 54c, 54d, 54e, 54f, 54g, 54h, 54i, 54j, 54k, 54l, 54m,54n, 54o, 54p, 58a, 58b, 58c, 58d, 58e, 58f, 58g, 58h, 58i, 58j, 58k,58l, 58m, 58n, 58o, 58p, 64, 68a, 68b, 68c, 68d, 68e, 68f, 68g, 68h,68i, 68j, 68k, 68l, 68m, 68n, 68o, 68p, 71a, 71b, 71c, 71d, 71e, 71f,71g, 71h, 71i, 71j, 71k, 71l, 71m, 71n, 71o, or 71p as described inEXAMPLE 26 except the wavelengths of excitation and emission areoptimized as determined by one skilled in the art before conducting theassay. In each case, the relative fluorescence intensity is then readand recorded to generate a standard curve which is directly proportionalto the quantity of dsDNA hybridization, or target sequence, under theseconditions.

EXAMPLE 28

Hybridization Assay for d(pT)9 and d(pA)9 Using Intercalator Compound 7,8, 50, 54a, 54b, 54c, 54d, 54e, 54f, 54g, 54h, 54i, 54j, 54k, 54l, 54m,54n, 54o, 54p, 58a, 58b, 58c, 58d, 58e, 58f, 58g, 58h, 58i, 58j, 58k,58l, 58m, 58n, 58o, 58p, 64, 68a, 68b, 68c, 68d, 68e, 68f, 68g, 68h,68i, 68j, 68k, 68l, 68m, 68n, 68o, 68p, 71a, 71b, 71c, 71d, 71e, 71f,71g, 71h, 71i, 71j, 71k, 71l, 71m, 71n, 71o, or 71p

By using the above described procedure in EXAMPLE 25, complementaryoligonucleotide d(pA)9 and d(pT)9 hybridization can be determined exceptthat the wavelengths are subtituted to optimal wavelengths for eachcompound as is determined by one skilled in the art. The describedprocedure is then followed using the optimal wavelengths as aredetermined.

EXAMPLE 29

Gel Electrophoresis Application Using Intercalator Compound 1, 2, 3, 25,26, 27, 28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b, 36b, 37b, 38b, 39b, 40b,41b, 42b, or 80

An agarose gel can be run to detect DNA using compound 1, 2, 3, 25, 26,27, 28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b, 36b, 37b, 38b, 39b, 40b,41b, 42b, or 80. Plasmid, pBR322, at 2.1 mg in 7 ml stock is incubatedat 37° C. for 1 hour with 1 ml of BAMH restriction enzyme with 2 ml 10×React2 Buffer and diluted to 20 ml total with 10 ml H₂ O. Alternatively,any other appropriate DNA sample is substituted for the above described"nicked" plasmid. This mixture is then used to prepare 3 stocks ofnicked pBR322 plasmid at 0.63 mg per 6 ml for each vial. Each of thesestocks is diluted further with H₂ O and 20% glycerol to final DNA stocksof 20 ng/ml, 800 pg/ml, 160 pg/ml, and 40 pg/ml with a 1:4 ratio of dyeto DNA base pairs in each for a total of 12 stocks. A 5 μl aliquot ofeach stock is loaded into 12 separate lanes in agarose gel andelectrophoresis is run for 30 minutes in 4 mM TRIS, pH8.2, with 0.01 mMEDTA buffer. The gel is then removed and photographed under exposure toU.V. light in a conventional gel box. Alternatively, the gel is scannedusing fluorescence confocal microscopy or charge coupled device imagingas a fluorescence detection or visualization method.

EXAMPLE 30

Protocol for Synthesis of Intercalator Activated Carboxymethyl StyreneMicroparticle Capture Reagent Using Intercalator Compound 25, 26, 27,29b, 35b, 38b, 50, 54a, 54c, 58a, 58c, 64, 68a, 68c, 71a, 71c, 80, 85,or 90

The synthesis of intercalator derivatized solid phase microparticle (MP)capture reagent can be accomplished by the following procedure:

A 45 aliquot of 0.275±μm microparticles (Seradyne, Indianapolis, Ind.)is placed in a 4 ml vial and the surfactant is exchanged out usingBio-Rex 501-D ion exchange mixed bed resin (Bio-Rad, Richmond, Calif.).After gentle shaking for 2 hours, the resin is filtered out from themixture by using a coarse fritted glass funnel equipped with a reducedpressure collection chamber. The sample is diluted to a concentration ofmp at 10% solids by weight. The total amount of equivalents of reactivecarboxylic acid is calculated from the titration specifications of thevendor. A stock solution of sulfo N-hydryoxysuccinimide (Pierce,Rockford, Ill.) is made at 11 mg/ml (20 mM) in H₂ O and a stock solutionof EDAC (Sigma Chemical Co., St. Louis, Mo.) at 10 mg/ml (5 mM) is madein H₂ O. Five equivalents of EDAC (290 μl stock) is added to thecarboxymicroparticle reaction mixture, followed by 5.0 equivalents ofsulfo N-hydryoxysuccinimide (330 μl stock). This mixture is allowed toincubate at room temperature for 2 hours and then a 2.0 molar equivalentof intercalator compound 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c, 58a,58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90 (4 mg) is added at aconcentration of 8 mg/400 μl, or 2.0 mg/100 μl in pH 8.0 0.1 N NaCl 0.1NPi phosphate buffer. N-hydryoxysuccinimide (Pierce) can be substitutedfor sulfo N-hydryoxysuccinimide if it is first dissolved in a stock ofDMF (Dimethyl formamide) and aliquoted as described above. Afterallowing 24 hours for complete reaction, the free dye is then removed bycentrifugation, removal of mother liquor, and resuspension for severalattempts until the solution went clear and no more dye is extracted fromthe samples. The purified capture reagent is then diluted to a stock of2-4% solids in H₂ O.

EXAMPLE 31

Solid Phase DNA Capture Using Solid Phase Derived from Compound 25, 26,27, 29b, 35b, 38b, 50, 54a, 54c, 58a, 58c, 64, 68a, 68c, 71a, 71c, 80,85, or 90

The capture of DNA onto solid phase can be performed usingderivatization methods as described in EXAMPLE 5 except substituting theappropriate solid phase synthesized from the respective intercalator 25,26 27, 29b, 35b, 38b, 50, 54a, 54c, 58a, 58c, 64, 68a, 68c, 71a, 71c,80, 85, or 90 as described in EXAMPLE 30.

Other appropriate solid phases such as carboxylated polystyrenemicroparticles or carboxylated magnetic microparticles can are also usedinstead of CM Sepharose.

EXAMPLE 32

Protocol for DNA Capture by Intercalator Modified Solid Phase ModifiedBy Compound 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c, 58a, 58c, 64, 68a,68c, 71a, 71c, 80, 85, or 90

DNA can be released from solid phase as described in EXAMPLE 6 exceptthat corresponding solid phase derived from intercalator compound 25,26, 27, 29b, 35b, 38b, 50, 54a, 54c, 58a, 58c, 64, 68a, 68c, 71a, 71c,80, 85, or 90 is used rather than the PTA 24 derivatized intercalator.

EXAMPLE 33

Fluorescence Staining in a Flow Cytometric Study of Chicken ErythrocyteNuclei (CEN)

Protocol: 50 μl of whole blood sample from two in-house donors and 3 μlof CEN suspension is added to 1.0 ml of pre-warmed at 40° C. WBC DILwithout and with the compound 1, 2, 3, 25, 26, 27, 28b, 29b, 30b, 31b,32b, 33b, 34b, 35b, 36b, 37b, 38, 39b, 40b, 41b, 42b, or 80 at 1 μg/mlconcentration, mixed, introduced to the FACScan™ and 20" readings isacquired. Chicken erythrocyte nuclei (CEN) is used to measure thebrightness of the FL3 staining (mean FL3 of CEN). The whole bloodsamples used is about 4-5 hours old.

EXAMPLE 34

Viability Dyes on the Coulter Elite Flow Cytometer Using Compound 1, 2,3, 25, 26, 27, 28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b, 36b, 37b, 38,39b, 40b, 41b, 42b, or 80

Cell Isolation Protocol: Each tube of ficol isolated cells can betreated as follows: PBS with 0.1% NaAzide and 1.0% albumin (Sigmacatalogue #1000-3) Ficol specific gravity 1.119 (Sigma Histopaguecatalogue #1119-1).

10 ml of whole blood (EDTA anticoagulant) is diluted with 10 ml of PBSW.Into 4, 15 ml conical bottom tubes, 5 ml of the diluted blood is layeredover 5 ml of ficol. The tubes is spun for 30 minutes at 400×G. Theinterface layer which contains the lymphocytes, monocytes, granulocytesand platelets is aspirated and washed once in 5 ml PBS, by centrifugingtubes at 300×G for 6 minutes. The cell pellet is resuspended in PBS,cells counted, and adjusted to 8.5×106 cells per ml.

Cell Staining Protocol:

Dye or Compound Solutions:

Compound 1, 2, 3, 25, 26, 27, 28b, 29b, 30b, 31b, 32b, 33b, 34b, 35b,36b, 37b, 38, 39b, 40b, 41b, 42b, or 80--Stock solution 10 μg/ml made bydissolving dye in PBS with 0.1% NaAzide.

Staining of compound 1, 2, 3, 25, 26, 27, 28b, 29b, 30b, 31b, 32b, 33b,34b, 35b, 36b, 37b, 38, 39b, 40b, 41b, 42b, or 80: In 12×75 mm tube,23.5 μl of cells is gently mixed with 76 μl of the compound stocksolution. After 20 seconds, the tube is placed on Elite flow cytometerand data collected.

Flow Cytometer Protocol: Cells analyzed on the Elite flow cytometer(Coulter Electronics, Inc.).

Samples can be excited with an argon laser at 488 nm and 15 mW of power.Data is gated on the basis of size and granularity to exclude red bloodcells, platelets and debris. The linear dye fluorescence of the gateddistribution is analyzed using unstained cells as a control. The percentpositive events (dead cells) and the mean fluorescence of the dead celldistribution is recorded.

EXAMPLE 35

Conjugates of Intercalator Derivatized Antibodies and IntercalatorDerivatized Alkaline Phosphatase Using Double Stranded Nucleic Acids asConjugation Templates

Complementary strands of oligonucleotides, sense and antisense 14 to20-mers, can be synthesized on a fully automated DNA synthesizer andpurified as described in Bioconjugate Chemistry, 4, pp. 94-102, (1993).The synthesis of the conjugate between the enzyme calf intestinalalkaline phosphatase and IgG can be accomplished as follows.

a) Derivatization of the Intercalator PTA 24 with 30 AtomHeterobifunctional Linker Arm,4-[(N-maleimidomethyl)tricaproamido]cyclohexane-1-carboxylate (SMTCC).

PTA 24, 0.100 g (2.3×10⁻⁴ mole), is dissolved in 5 ml of 50% aqueous DMF(dimethylformamide). 30 atom heterobifunctional linker,4-[(N-maleimidomethyl)tricaproamido]cyclohexane-1carboxylate (SMTCC),0.157 g, (2.3×10⁻⁴ mole) synthesized as described in Bieniarz et al.,U.S. Pat. Nos. 4,994,385, 5,002,883, 5,053,520, and 5,063,109 isdissolved in 3 ml of DMF and is added in one portion to the solution andstirred over 24 h at ambient temperature. The resulting maleimidederivatized PTA 24 contains is purified on silica gel column using 10%methanolic acetone as eluent.

b) Derivatization of the Calf Intestinal Alkaline Phosphatase withIminothiolane.

Calf intestinal alkaline phosphatase, 6 mg (4×10⁻⁹ mole) in 1 ml of PBSbuffer is thiolated by treatment with a 450-fold molar excess ofiminothiolane (164 ml of a 15 mg/ml solution in PBS buffer) for 30 minat ambient temperature. Excess reagent is removed by gel filtration witha Sephadex G-25 column (1×45 cm) equilibrated with PBS buffer. Thefractions containing the derivatized enzyme are pooled and theconcentration of the enzyme in the pool is calculated by absorbance at280 nm.

c) Conjugation of the SMTCC Derivatized PTA 24 to the Thiolated CalfIntestinal Alkaline Phosphatase from b).

The thiolated calf intestinal alkaline phosphatase from part b) andmaleimide derivatized PTA 24 from part a) are combined at molar ratio1:1 and incubated 18 h at 5° C. Unreacted thiol groups are capped byaddition of 5 mM N-ethylmaleimide to 0.3 mM final concentration followedby 1 h incubation at ambient temperature.

d) Derivatization of IgG with thiolates using site-specificfunctionalization of the Fc region of IgG. Site-specific introduction ofthiolates into Fc region of IgG is accomplished as described in Bieniarzet al., U.S. Pat. No. 5,191,066. In that way, between 4 and 10 thiolatesare introduced into Fc region of the IgG.

e) Derivatization of the Fc Thiolated IgG from part d) with maleimidederivatized PTA 24 from part a).

The Fc thiolated IgG from part d) and maleimide derivatized PTA 24 frompart a) are combined at molar ratio of 1:5 of thiolated IgG to SMTCCderivatized PTA 24. The solution is incubated in phosphate buffer pH 7.0for 18 h at 5° C. and the conjugate is purified on gel filtration columnSephadex G-25. Fractions containing proteins are pooled and concentratedusing Amicon concentrator. Protein content of the final solution isestablished using Coomassie Dye Binding Assay, from Pierce Company.

f) Conjugation of the PTA 24 derivatized IgG from part e) to the PTA 24derivatized calf intestinal alkaline phosphatase from part c) usingdouble stranded 20-mer oligonucleotide composed of the complementarystrands of the oligos.

Complementary strands of oligonucleotides are hybridized and examined asdescribed in Bioconjugate Chemistry, 4, pp. 94-102, (1993). PTA 24derivatized IgG from part e), PTA 24 derivatized calf intestinalalkaline phosphatase and double stranded oligonucleotide 20-mer used asthe conjugation template are incubated overnight at pH 7.0 for 18 h inmolar ratios of 1:1:1 at ambient temperature. The conjugate is filteredthrough a gel filtration column G-25. The fractions are assayed foralkaline phosphatase activity using fluorogenic substrate4-methylumbelliferyl phosphate, and for the antibody activity using theantiidiotypic IgG labelled with fluorescein.

The conjugate is also examined by gel filtration HPLC columns.

EXAMPLE 36

Conjugation of Intercalator Derivatized Liposome to a Double StrandedDNA

Preparation of the liposomes is carried as described in Fiechtner etal., U.S. Pat. No. 4,912,208. These liposomes display on their surfaceprimary amines because they are prepared fromdiphosphatidiylethanolamine lipids. The preparation of the liposomes iscarried in presence of membrane impermeable fluorescent dyes disclosedin U.S. Pat. No. 4,912,208. Thus, the molecules of the fluorescent dyesare substantially at very high, self quenching concentrations inside theliposomes and consequently they are nonfluorescent. The surface aminesof the liposomes are derivatized with thiolates by essentially the samemethod used to introduce thiolates into alkaline phosphatase describedin the EXAMPLE 35. PTA 24 and other intercalators disclosed in thepreceding examples are derivatized with maleimides, utilizing SMTCCreagent, as described in EXAMPLE 35. Thiolate derivatized liposomes andmaleimide derivatized PTA 24 or other intercalators are incubatedtogether in a vessel under conditions esentially identical to the onesdescribed in EXAMPLE 35. The fluorescent dye containing liposomesderivatized with multiple intercalators are incubated at pH ranging from5 to 9, preferably 7.0 with the sample containing small concentrationsof the double stranded DNA coated or immobilized on a solid phase. Theincubation is followed by wash and addition of detergent. Theconcomitant lysis of the liposome membrane results in spilling of thefluorogenic dye, dequenching of the fluorescence and appearance ofsignal.

Alternatively, the probe multimeric single stranded DNA may beimmobilized on a solid phase; the patient sample suspected to contain acomplementary single stranded DNA is incubated in presence of the probe;the liposome-intercalator is brought in contact with the hybridizedsample and the contents of the vessel are incubated and washed severaltimes to remove excess of the PTA 24 derivatized liposomes. If the probefinds a complementary sequence in the patient sample, the addition ofthe detergent results in lysis of the liposomes and signal. If howeverthe probe and patient sample DNA strands are not complementary, nodouble stranded DNA is present and substantially all the PTA 24derivatized liposome is washed away from the solid phase, resulting inno signal.

EXAMPLE 37

Detection of DNA Using A Fluorescent Intercalator Conjugate Derived FromIntercalator Compound 24, 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c, 58a,58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90

The synthesis of intercalator maleimide functionalized intercalator canbe effected by the procedure as already described in EXAMPLE 35 exceptthat the intercalator compound 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c,58a, 58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90 is used in place of PTA24. An appropriate signal generating entity such as phycoerythrin orallophycocyanine is covalently attached to this maleimide derivatizedintercalator through a thiolate on the thiolated protein prepared asdescribed in EXAMPLE 35. After allowing time for binding of theintercalator portion of the conjugate to the immobilized ds-DNA moleculeand washing away the unbound conjugate, then double stranded DNA isdetected on the solid phase or other immobilized entity using thefluorescence of the phycoerythrin that is localized by the binding ofthe intercalator portion to the double stranded DNA molecule. Thefluorescence emission is read at approximately 580 nm using methodsknown to those skilled in the art while exciting the fluorescent proteinat a wavelength that is appropriate for efficient excitation as isdetermined by one skilled in the art.

EXAMPLE 38

Detection of DNA Using an Enzyme Intercalator Conjugate Derived fromIntercalator Compound 24, 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c, 58a,58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90

The synthesis of intercalator maleimide functionalized intercalator canbe effected by the procedure as already described in EXAMPLE 35 exceptthat the intercalator compound 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c,58a, 58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90 is used in place of PTA24. An appropriate signal generating entity such as alkalinephosphatase, β-galactosidase, esterase, or β-lactamase is covalentlyattached to this maleimide derivatized intercalator through the thiolateof the thiolated protein that is prepared as described in EXAMPLE 35.After allowing time for binding of the intercalator portion of theconjugate to the immobilized ds-DNA molecule and washing away theunbound conjugate, then double stranded DNA is detected on the solidphase or other immobilized entity such as a colloid or microparticle byfluorescence or chemiluminscence afforded by the turn-over of anon-fluorescent or non-chemiluminescent substrate of the appropriateenzyme to a fluorescent or chemiluminescent entity respectively as isdetermined by one skilled in the art. The fluorescence emission orchemiluminescence is then read at at the appropriate wavelengths byusing standard methods of fluorescence excitation or detection ofchemiluminescence respectively that are known to those skilled in theart.

EXAMPLE 39

Detection of DNA Using an Intercalator--Chemiluminophore ConjugateDerived from Intercalator Compound 24, 25, 26, 27, 29b, 35b, 38b, 50,54a, 54c, 58a, 58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90

The synthesis of intercalator maleimide functionalized intercalator canbe effected by the procedure as already described in EXAMPLE 35 exceptthat the intercalator compound 25, 26, 27, 29b, 35b, 38b, 50, 54a, 54c,58a, 58c, 64, 68a, 68c, 71a, 71c, 80, 85, or 90 is used in place of PTA24. An appropriate activatable chemiluminescent signal generating entitysuch as acridinium sulfonamide is covalently attached to thisintercalator by thiolate of the chemiluminescent entity that is preparedso as to be reactive towards the maleimide such as can be devised by oneskilled in the art. After binding of ds-DNA with theintercalator--chemiluminophore conjugate, the excess or unboundconjugate is washed away and double stranded DNA is then detected on thesolid phase or other immobilized entity by direct chemiluminscencetriggered by the addition of an appropriate activation reagent such ashydrogen peroxide leading to a chemiluminescent entity andchemiluminescence is then read at at the appropriate wavelengths byusing standard methods of detection of chemiluminescence such as areknown to those skilled in the art.

EXAMPLE 40

Synthesis of Intercalator Derivatized Dendrimer

Intercalator derivatized dendrimer can be synthesized from DendriticPolymers obtained from PolySciences. The intercalator bromideintermediates 22, 49, 58, 63, 71, 83, or 88 is used to alkylate theamines on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th generationdendrimer by heating in dimethyl formamide or other appropriate organicsolvent to between 30-90° C. for 0.25-72 hours. The product is thenpurified in the case of when intercalator compound 58, 63, or 71 is usedby using size exclusion chromatography on a G-25 SephadexTM column inwater or an appropriate aqueous buffer and the intercalator derivatizeddendrimer is obtained. In the case of intercalator compound 22, 83, or88, the column in G-25 is run in water and the protected intercalatorderivatized dendrimer is then isolated. This material is then subjectedto heating to 90° C. for 2-4 hours in 4N HCl to hydrolyze the aromaticamine protecting groups and then the solution is neutralized to pH 4-10and a G-25 column is run in an appropriate buffer on the final productto obtain pure intercalator derivatized dendrimer.

The various literature references on the general synthesis given in theexamples above are hereby incorporated by reference.

Although the present invention and its advantages have been described indetail, those skilled in the art should understand that they can makevarious changes, substitutions and alterations herein without departingfrom the spirit and scope of the invention as defined by the appendedclaims.

We claim:
 1. A compound having a formula I-T_(m), m is an integer of 1or 2, where T is 2, T is the same or different from one another, I isselected from the group consisting of: ##STR12## T is selected from thegroup consisting of: ##STR13##
 2. A compound having the formula whereinA⁻ is an acceptable monvalent counter anion.
 3. The compound of claim 2,wherein said A⁻ is selected from the group consisting of chloride,bromide and iodide.
 4. A compound having the formula ##STR14## whereinA⁻ is an acceptable monvalent counter anion.
 5. The compound of claim 4,wherein said A⁻ is selected from the group consisting of chloride,bromide and iodide.
 6. A compound having the formula ##STR15## whereinA⁻ is an acceptable monvalent counter anion.
 7. The compound of claim 6,wherein said A⁻ is selected from the group consisting of chloride,bromide and iodide.
 8. A compound having the formula ##STR16## whereinA⁻ is an acceptable monvalent counter anion.
 9. The compound of claim 8,wherein said A⁻ is selected from the group consisting of chloride,bromide and iodide.